Structure-Activity Relationship Studies in Substituted Sulfamoyl Benzamidothiazoles that Prolong NF-κB Activation

Abstract

In the face of emerging infectious diseases, there remains an unmet need for vaccine development where adjuvants that enhance immune responses to pathogenic antigens are highly desired. Using high-throughput screens with a cell based nuclear factor κB (NF-κB) reporter assay, we identified a sulfamoyl benzamidothiazole bearing compound 1 that demonstrated a sustained activation of NF-κB after a primary stimulus with a Toll-like receptor (TLR)-4 agonist, lipopolysaccharide (LPS). Here, we explore systematic structure-activity relationship (SAR) studies on compound 1 that indicated the sites on the scaffold that tolerated modification and yielded more potent compounds compared to 1. The selected analogs enhanced release of immunostimulatory cytokines in the human monocytic cell line THP-1 cells and murine primary dendritic cells. In murine vaccination studies, select compounds were used as co-adjuvants in combination with the Food and Drug Administration approved TLR-4 agonistic adjuvant, monophosphoryl lipid A (MPLA) that showed significant enhancement in antigen-specific antibody titers compared to MPLA alone. Additionally, our SAR studies led to identification of a photoaffinity probe which will aid the target identification and mechanism of action studies in future.

Graphical Abstract

Introduction:

In the face of emerging infectious diseases where viruses continue to mutate to escape host immunity or emergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there remains an unmet need for vaccine development.^1-5 Vaccination is a cost-effective healthcare strategy to prevent infections and has been successful in reducing the incidence of infectious diseases including diphtheria, pertussis, measles, polio, rotavirus, rubella and tetanus. However, many vaccines require repeated injections and boosters for protective immunity and yet do not confer extended protection. For vaccines against diseases such as influenza, a broader immunoprotection would aide in the defense against viruses that undergo antigenic drift and shift, which are the potential cause of epidemics and pandemics, respectively.^6-8 To address such needs, novel vaccine adjuvants are being investigated. Many candidate adjuvants are components of bacterial or viral origin, or synthetic ligands for pattern recognition receptors (PRRs) including Toll-like receptors (TLR) −2, −4, −7, −8, and −9, nucleotide-binding oligomerization domain-like receptors (NLRs), and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)^9-18 To date the U.S. Food and Drug Administration (FDA) has approved a TLR-4 agonist monophosphoryl Lipid A (MPLA)¹⁹, TLR-9 agonist CpG 1018,²⁰ alum and squalene based adjuvants for human use. Although these adjuvants were approved for use in specific vaccines, the need for improving adjuvant activity led to our focus for the discovery of co-adjuvants, that promote efficacy of a known adjuvant.²¹ For example, AS04 (Adjuvant System 04), consists of MPLA and alum, and is approved in a hepatitis B vaccine, Fendrix^®, and a human papillomavirus vaccine, Cervarix^®.^22-25

Our efforts towards identifying co-adjuvants focused on compounds that may enhance the NF-κB and/or interferon (IFN) stimulating response element (ISRE) activation induced by a TLR-4 agonist or IFN-α, respectively.^26,27 These compounds were clustered into multiple chemotypes such as pyrimido[5,4-b]indoles that had distinct structural requirements for prolonging NF-κB activation compared to a set of compounds that induced NF-κB activation as TLR-4 agonists.^26,28 Additional chemotypes included compounds that enhanced both NF-κB and ISRE signaling such as 4H-chromene-3-carbonitriles which were found to be good adjuvant for cancer immunotherapy and bis-aryl sulfonamides that were found to enhance adjuvanticity of TLR-4 agonists.²⁹ All these compounds mentioned above were identified via cell-based phenotypic assays, so we aim to first analyze the SAR studies to apply and utilize them to understand mechanism of action and target receptor identification in the future. Here we report the structure-activity relationship (SAR) studies in one such chemotype bearing a substituted sulfamoyl benzamidothiazole structure that is specific towards enhancing the NF-κB activation. These SAR studies are aimed at identifying more potent compounds as well as utilizing SAR understanding towards design and synthesis of an affinity probe analog.

Results and Discussion:

Preliminary SAR studies based on HTS data of the screening library compounds:

The library used in the HTS included 248 compounds that belonged to the substituted sulfamoyl benzamidothiazole chemotype. These 248 compounds provided a preliminary SAR understanding which is important from the perspective of further designing novel and potent compounds as it guides drug design. The detailed scaffold classification for these 248 compounds and the potency in enhancing NF-κB signaling activity in the presence of LPS as part of HTS is presented in Supporting Information Fig. S1.

The subset of 21 compounds that were identified to be active in enhancing NF-κB activity in the presence of LPS after 12 hours in HTS were then subjected to confirmatory kinetic screening for NF-κB enhancement activity at 5h and 12h in the presence of LPS. A scatter plot showing the activation data for these compounds in the cell-based NF-κB FRET assay for 12h (Y axis) and 5h (X-axis) incubation is shown in Supporting Information Fig. S2. Unlike previously identified chemotypes that enhanced NF-κB activity which could be clearly classified into 4 groups based on the 4 quadrants of the kinetic plot;²⁶ here this series of compounds did not statistically affect the activity at 5h but significantly enhanced the NF-κB activity by LPS at 12h. Of all these compounds, compound 1 was found to be the most potent (Supporting Information Fig. S2). The structure of compound 1 bearing a 2,5-dimethylphenyl substituent on the 4 position of the thiazole ring and benzoyl-4-(sulfonylpiperidine) substituent connected at the 2-amino position of the thiazole ring is shown in Fig. 1. Compound 1 was then subjected to further SAR studies with an aim to not only identify more potent compounds but also to understand the positions on the scaffold that could tolerate modification. We systematically divided the structure into 6 different sites of modification (A, B, C, D, E and F) as shown in Fig. 1. Site A was assigned the 4-substituent of the thiazole ring, site B was the thiazole ring itself, site C was the central amide bond in the structure, site D was the benzene ring bearing the 1,4-carboxyl and the sulfamoyl groups, site E was the sulfonamide functional group and site F was the amine substituent of the sulfonamide group. SAR studies on compound 1 were approached by modifying one site at a time.

Figure 1.

Structure and sites of modification of compound 1. The structure of compound 1 was reduced to six constituent components (A-F) based on the substituents and the synthetic strategy as shown in alternate blue and black colors.

Syntheses of analogs of compound 1 and SAR studies:

We began with the synthesis of compound 1 to understand the chemistry that may be utilized in the syntheses of analogs. The synthesis of compound 1 required the two advanced synthons (compounds 4 and 8a) as shown in Scheme 1. Compound 4 was obtained by formation of the sulfonamide bond by reaction of compounds 2 and 3. Compound 8a was obtained by first activation of the alpha carbon by bromination of the 2,5-dimethyl acetophenone (6a) to obtain compound 7a, followed by fusing the activated ketone with thiourea. These two synthons were coupled using the HATU reagent via amide bond to yield compound 1. A similar strategy was used for the synthesis of several other SAR analogs where the advanced synthon 4 was used for syntheses of site A and site B modified compounds while synthon 8a was utilized for syntheses of site D, E and F modified compounds.

Scheme 1. Syntheses of compound 1 and site A modified compounds.

Reagents and conditions: i. (a) Et₃N, CH₂Cl₂, (b) LiOH, MeOH/THF/H₂O; ii. (a) N,O-dimethylhydroxylamine hydrochloride, HATU, Et₃N, DMF, (b) MeMgBr, THF; iii. Br₂, CHCl₃; iv. thiourea, EtOH, 100 °C; v. HATU, Et₃N, DMF, 45 °C; vi. N,N-diisopropylamine, n-BuLi, THF; vii. (a) RB(OH)₂, Pd(PPh₃)₄, Na₂CO₃, DMF/H₂O; viii. 4N HCl/dioxane; ix. (a) TMS acetylene, Cul, Pd(PPh₃)₄, DMF (b) TBAF, THF; x. Pd/C, H₂ (50psi), MeOH.

Site A modified analogs:

With the preliminary SAR analysis in mind, we began the further SAR studies with site A modified analogs by first eliminating one methyl unit at a time of the 2,5-dimethylphenyl substituent to obtain 2-methyl and 3-methyl substituted compounds 12b and 12c, respectively (Scheme 1). We evaluated these compounds for NF-κB activation to evaluate the NF-κB inducing effects at longer incubation times (20h). NF-κB activation was measured by THP1-Blue™ NF-κB Cells, that secreted alkaline phosphatase (SEAP) in response to NF-κB. Both of the des-methyl compounds were found to be inactive suggesting a bis-substituted phenyl may be necessary for activity (Table 1). Thus, for the next two compounds, we exchanged the methyl group at each position with a bromo substituent, aiming to verify the functional group compatibility and to use it as a reactive intermediate for further aromatic nucleophilic substitution type reactions. 2-Bromo-5-methyl substituted compound 12d and 5-bromo-2-methyl substituted compound 12e were then synthesized as shown in Scheme 1. Since the corresponding acetophenone reagents (6d and 6e) for making these two compounds were not commercially available, we had to synthesize them from carboxylic acid (5d and 5e) precursors by a Weinreb amide synthesis, followed by Grignard reaction with methyl magnesium bromide. Both these bromo substituted analogs were active suggesting these could be utilized for further modification (Table 1). Next, we spaced the 2,5-dimethyl substituent away from the thiazole ring with a methylene unit to obtain compound 12f. However, the compound was inactive suggesting a bi-aryl conformation may be necessary for activity (Table 1). In the next set of compounds, we probed if the phenyl ring could be replaced with nonpolar aliphatic substituents of small and bulky sizes. Thus, 4-thiazole substituted methyl (12g), ethyl (12h), propyl (12i), iso-pentyl (12j) and tert-butyl (12k) analogs were synthesized. None of these molecules had any NF-κB activity suggesting that a bis-substituted phenyl group is necessary for activity (Table 1).

Table 1.

Bioactivity data for all synthesized compounds.

Compound number	Modified Site	NF-κB SEAPa Compound alone		MTTb Compound alone
		% Activation	SEM	% Viability	SEM
1	--	200	-	103	3.3
12b	A	100	0.2	98	1.8
12c	A	100	0.3	98	1.7
12d	A	254	12.2	98	3.0
12e	A	207	15.9	91	5.8
12f	A	101	0.2	100	3.5
12g	A	100	0.3	108	5.5
12h	A	100	0.7	120	7.9
12i	A	100	0.6	115	6.6
12j	A	101	0.5	116	5.5
12k	A	99	0.4	113	7.5
12l	A	179	12.5	103	5.4
12m	A	98	0.4	102	3.3
12n	A	98	0.5	98	4.9
12o	A	97	0.8	91	4.2
12p	A	185	4.5	100	6.1
12q	A	198	7.9	93	4.4
12r	A	109	3.2	89	4.3
12s	A	114	7.8	95	4.6
12t	A	101	0.6	92	6.0
12u	A	203	5.7	102	1.2
18a	B	99	0.9	51	0.6
18b	B	100	1.2	94	1.7
18c	B	100	0.8	93	2.9
18d	B	100	0.8	93	2.0
18e	B	99	0.4	101	1.7
18f	B	100	0.2	90	5.7
18g	B	99	0.5	94	5.0
18h	B	99	0.4	89	2.4
18i	B	99	0.4	98	4.0
18j	B	100	1.2	106	3.1
18k	B	103	0.9	102	5.9
18l	B	102	1.0	104	3.6
18m	B	159	7.2	97	7.8
18n	B	133	3.2	84	3.0
18o	B	118	5.3	111	6.2
18p	B	264	14.7	103	3.1
18q	B	393	12.2	106	3.9
18r	B	179	12.2	104	3.1
18s	B	109	3.7	129	7.7
18t	B	117	4.3	108	3.2
34a	C	99	0.9	85	2.7
34b	C	99	0.8	91	1.7
34c	C	101	1.0	97	3.8
34d	C	100	0.3	102	4.6
42a	D	99	0.7	94	1.2
42b	D	100	1.1	89	2.0
42c	D	103	1.6	97	5.8
42d	D	223	8.9	88	2.3
42e	D	209	9.2	90	1.7
42f	D	115	2.5	93	1.2
42g	D	99	1.0	99	2.8
42h	D	127	4.0	87	2.3
42i	D	120	4.6	96	2.5
42j	D	99	1.9	99	2.5
42k	D	125	2.5	92	2.5
45a	E	100	0.5	85	2.5
45b	E	100	0.8	82	2.2
45c	E	99	0.6	88	0.8
45d	E	100	0.5	88	2.9
51	C,E	103	1.1	69	6.4
54a	F	183	11.0	104	15.7
54b	F	167	19.0	96	4.3
54c	F	102	0.9	101	6.9
54d	F	105	2.5	81	2.3
54e	F	100	0.9	91	4.0
54f	F	101	0.8	132	3.6
54g	F	201	4.7	117	6.6
54h	F	517	34.8	111	7.1
54i	F	101	0.7	121	0.3
54j	F	101	0.5	114	4.9
54k	F	100	0.7	80	13.5
55	A,F	191	4.4	97	2.9
57	B,F	112	3.0	102	1.9
60	A,B	202	3.4	95	2.4
61	A,B,F	111	0.7	97	2.4

^aThe % activation values in NF-κB SEAP assay for compounds were two point normalized between compound 1 as 200% and Vehicle (0.5% DMSO) as 100%. The mean SEAP response in NF-κB assay for compound 1 and DMSO was 0.4 ± 0.02 and 0.033 ± 0.001 μg/mL, respectively.

^bThe % viability values for compounds in MTT assay was normalized to DMSO as 100%. The mean OD value at 405 nm for DMSO was 1.14 ± 0.01. All the compounds were evaluated at 5μM concentration. All raw values used for normalization are represented as mean ± SEM.

Thus, we then varied the substitution pattern of the two methyl groups on the phenyl to identify if any pattern correlated with activity. Molecules bearing all the different bis-methyl substituted configurations at ortho (o), meta (m), and para (p) positions were synthesized. This included o,m (2,3-dimethyl, 12l), o,p (2,4-dimethyl, 12m), m,p (3,4-dimethyl, 12n), m,m (3,5-dimethyl, 12o), and o,o (2,6-dimethyl, 12p) analogs as shown in Scheme 1. In order to evaluate an alternate route to synthesize these analogs from the perspective of improving synthetic yields and accessing aryl compounds for which the ketone synthon may not be available, we began with tert-butyl (5-bromothiazol-2-yl)carbamate (9), performed a halogen dance reaction³⁰ to migrate the bromo to position 4 on the thiazole ring and then used it as a common precursor for different Suzuki coupling reactions, followed by N-Boc removal using HCl/dioxane and a HATU assisted coupling reaction with 4 to obtain the differently substituted bis-methyl analogs 12l-12o. Since our attempts to perform the Suzuki coupling reaction with 2,6-dimethylphenylboronic acid to obtain compound 12p failed, likely due to steric hindrance, we used the ketone precursor 6p to obtain the bis ortho analog 12p. Of all these compounds, only the ortho and meta substitution bearing compounds 12l and 12p retained activity while the others were inactive (Table 1). Notably, compound 1 was o,m (2,5-dimethyl) substituted suggesting that substitution closer to the thiazole ring is tolerated while expansion on the ring at the para position results in loss of activity. Based on this observation, we elected to expand on the o,m position on the same side of the phenyl ring to obtain a α-naphthyl analog 12q, which was found to be equally potent, thus justifying our hypothesis (Table 1). Encouraged by the acceptance of steric bulk around the thiazole ring, we elected to lock the conformation of the phenyl and the thiazole ring by forming a 5 or 6 membered ring connecting the ortho position of phenyl ring to position 5 of the thiazole to obtain compound 12r and 12s, respectively. However, both these compounds were weakly active suggesting the possibility of a different conformation for optimal activity (Table 1). We then utilized our existing SAR findings to derivatize the bromo to expand at the ortho and meta positions on the phenyl ring. Our attempts to displace the bromo substituent with an ethynyl unit at the ortho position of 12d failed, but we were able to obtain the corresponding ethynyl substituted compound 12t using a Sonogashira coupling reaction. This analog was further reduced using catalytic hydrogenation to obtain the chain expanded 5-ethyl-2-methyl analog 12u. While the ethynyl analog 12t was inactive, releasing the rigidity of the carbon-carbon bond as in 12u regained the NF-κB activity (Table 1).

Site B modified analogs:

All the compounds explored in the HTS contained a thiazole ring and most of them were modified at position 4 on the thiazole with very few compounds representing 5-substituted thiazole analogs (Supporting Information Fig. S1). Thus, we were interested to explore alternate heterocyclic replacement of thiazole as well as compounds modified at the 5-position on the thiazole ring. We synthesized imidazole analog 18a, different bioisosteric pyridine analogs including 2-amino-6-aryl (18b), 2-amino-5-aryl (18c) and 3-amino-6-aryl (18d) substituted pyridine analogs. In addition, flipping the thiazole moiety yielded 5-phenyl substituted compound 18e and addition of another nitrogen atom at position 5 yielded thiadiazole analog 18f (Scheme 2). However, none of these molecules that replaced the thiazole functionality was active, suggesting that thiazole may be involved in key interactions with the receptor component necessary for activity (Table 1). Since thiazole was indispensable, we decided to probe further by substituting at position 5 on the ring. Bromination of compound 8a using N-bromosuccinimide allowed for the introduction of 5-bromo on the thiazole. The 5-bromo was displaced using different reagents to obtain substituted amine analogs 18g and 18h, Suzuki reaction derived phenyl analog 18i and furan analog 18j. Furthermore, ethanethiol and ethoxide displaced the 5-bromo to obtain corresponding analogs 18k and 18l, respectively. All of these compounds were completely inactive except for the ethoxy analog 18l, which showed partial activity suggesting that linear chains may be tolerated (Table 1). Thus, we first synthesized a 5-methyl analog 18m using the chemistry shown in Scheme 2 as above. The 5-bromo-2-aminothaizole was amide-linked to obtain the bromo analog (18n) of compound 1, which was derivatized to obtain a series on carboalkyl chains including 2-carbon, 4-carbon, and 5-carbon alkynyls 18o, 18p, and 18q, and their corresponding alkyl derivatives 18r, 18s, and 18t, respectively, obtained by catalytic hydrogenation of the alkynes. As hypothesized, these compounds were potent in inducing NF-κB activity and we saw a chain length dependent activity trend as shown in Fig. 2 (Table 1). The smaller alkyl chain analogs 18r and 18s retained activity while the higher homologs showed dramatic loss in activity. In contrast, the ethynyl 18o had weak activity while higher chain homologs 18p and 18q were very potent surpassing the activity of compound 1 when tested alone (Fig. 2). These data, especially the potency of 5-alkyne substituted thiazole compounds suggested the presence of a hydrophobic pocket with the possible involvement of π-π interaction in the target receptor. Another possible hypothesis for the observed difference in potency could be the different orientation of the substituent attached to an alkyne or an ethyl group as observed with the enhanced potency of ponatinib where the ethynyl group functions as linker to span the bulky residues on the target protein.³¹ Overall, site B modification suggested the necessity to preserve the thiazole ring, but showed that NF-κB activity can be enhanced with compounds bearing 5-alkyne substitution.

Scheme 2. Syntheses of site B modified compounds.

Reagents and conditions: i. (a) Br₂, CHCl3, (b) 1-acetylguanidine, DMF; ii. 3M HCl, MeOH, 55 °C; iii. 4, HATU, Et₃N, DMF, 45 °C; iv. Pd(PPh₃)₄, Na₂CO₃, DMF/H₂O; v. (a) 9, Pd(PPh₃)₄, Na₂CO₃, DMF/H₂O, (b) 4N HCl/dioxane; vi. thiosemicarbazide, POCl₃, 105 °C; vii. N-bromosuccinimide, CH₂Cl₂; viii. For compound 26g: Pd(dppf)Cl₂, Et₂NH, dioxane, for compound 26h: piperidine, NaH, DMF, for compounds 26i-j: Y-B(OH)₂, Pd(PPh₃)₄, Na₂CO₃, DMF/H₂O; for compound 26k: NaSEt, DMF, for compound 26l: NaOEt, EtOH; ix. (a) Br₂, CHCl₃, (b) thiourea, EtOH, 100 °C; x. For compound 18o: (a) TMS acetylene, Pd(PPh₃)₄, Cul, Et₂NH, (b) TBAF, THF, For compounds 18p-q: RH, Pd(PPh₃)₄, Cul, Et₂NH; xi. Pd/C, H₂ (50 psi), MeOH.

Figure 2.

Chain length dependent NF-κB activity profile in 5-thiazolyl site B modified analogs. Differential activity profiles were observed for alkane v/s alkyne bearing analogs at the 5-thiazolyl position of compound 1. Chain length (n) dependent reduction in activity was observed for 5-thiazolyl modified alkanes (green squares) but an inverse activity relationship was observed with corresponding alkynes (blue circles). The % activation values in NF-κB induction assays were two-point normalizedor DMSO (0.5%) as 100% (grey dotted line) and compound 1 as 200% (grey dotted line). The mean SEAP response in NF-κB assay for compound 1 and DMSO was 0.4 ± 0.02 and 0.033 ± 0.001 μg/mL, respectively. The relative reduction in the activity for terminal alkane compounds was significantly greater than the terminal alkynes for the same chain length suggesting involvement of π-π interactions. Alkyne derivatives are shown in blue circles and alkane derivative activity is shown in green squares. Data are presented as mean ± SD. **p<0.01 and *p<0.05 for alkyne bearing compounds compared to alkane bearing compounds for the same chain length using one-way ANOVA followed by Kruskal-Wallis test.

Site C modified analogs:

Next, site C modified compounds required us to make new synthons as the amide linking synthetic strategy utilized so far could not be used since we wanted to evaluate the effect of changing this amide linkage. Thus, an amine linked analog 34a and an inverse amide linked compound 34b were synthesized as shown in Scheme 3. The alkylation of the amide nitrogen yielded N-methyl analog 34c and N-propargyl analog 34d. The importance of the hydrogen bond forming capabilities and positioning of the amide link was evident as all these site C modified analogs were inactive except the inverse amide linked compound 34b that retained only weak activity (Table 1).

Scheme 3. Syntheses of site C modified compounds.

Reagents and conditions: i. K₂CO₃, DMF; ii. Pd/C, H₂ (50 psi), MeOH; iii. (a) Br₂, CHCl₃, (b) NaSCN, EtOH; iv. HBr/AcOH, 130 °C; v. Pd(OAc)₂, dioxane; vi. (a) Br₂, CHCl₃, (b) ethylthioxamate, EtOH, 120 °C; vii. LiOH, MeOH, THF, H₂O; viii. HATU, Et₃N, DMF; ix. for compound 34c: iodomethane, NaH, DMF, for compound 34d: propargyl bromide, NaH, DMF.

Site D modified analogs:

Site D modification included bio-isosteric replacement of the benzene ring with pyridine to obtain compounds 42a and 42b and thiophene to obtain compound 42c (Scheme 4). However, these bioisosteres of the benzene ring led to loss of activity, so we prepared the next set of substituted benzene analogs. These included addition of a small group on the position next to sulfonamide (position 3) such as 3-chloro (42d), 3-bromo (42e), 3-nitro (42f), and 3-methoxy (42g). Also, the 3-nitro group on compound 42f was reduced to obtain the 3-amino analog 42k. While the 3-chloro and 3-bromo substituted compounds retained NF-κB activity, compounds containing the hydrogen bond forming substituents including 3-nitro, 3-methoxy and 3-amino showed a decline in activity. This drop suggested the need for hydrophobic components on the benzene ring. We queried the importance of hydrophobicity of the substituent on the benzene ring and whether the position on the ring matters as well. So, we synthesized the next set of compounds with substituents on the position next to the carboxamide (position 2) including 2-chloro (42h), 2-bromo (42i) and 2-methoxy (42j). The NF-κB activity significantly dropped for these analogs suggesting the presence of a small hydrophobic pocket on the receptor at position 3 of the chemotype.

Scheme 4. Syntheses of site D modified compounds.

Reagents and conditions: i. For compounds 41a-b: (a) piperidine, K₂CO₃ DMF, (b) LiOH, MeOH, THF, H₂O; For compounds 41c-j: when R=-CH₃ (a) piperidine, Et₃N, CH₂Cl₂ (b) LiOH, MeOH, THF, H₂O; when R=H (a) piperidine, K₂CO₃, DMF, ii. 8a, Et₃N, HATU, DMF, 45 °C: iii. Pd/C, H₂ (50psi), MeOH.

Site E modified analogs:

Next, site E modification involved changing the sulfonamide bond. This included elimination of hydrophilic interaction capable atoms like the sulfonamide nitrogen and sulfone oxygens to obtain thioether analog 45a, which was oxidized to yield sulfone analog 45b. Replacement of sulfonamide with carboxamide at position 4 and 3 provided compounds 45c and 45d, respectively, as shown in Scheme 5. Surprisingly, none of these modifications at site E was tolerated (Table 1). Overall, the SAR studies suggested that the sulfamoyl benzamide thiazole component consisting of sites B, C, D and E were critical for activity and may be the core pharmacophore. Thus, for curiosity we swapped the carboxamide bond with the sulfonamide bond to obtain compound 51 (Scheme 6) which was also found to be inactive (Table 1).

Scheme 5. Syntheses of site E modified compounds.

Reagents and conditions: i. K₂CO₃, DMF, 45 °C; ii. 8a, Et₃N, HATU, DMF, 45 °C; iii. 3-chloroperoxybenzoic acid; iv. HATU, Et₃N, DMF.

Scheme 6. Syntheses of substituent swap analog.

Reagents and conditions: i. (a) Et₃N, CH₂Cl₂, (b) LiOH, MeOH, THF, H₂O; ii. piperidine, HATU, Et₃N, DMF.

Site F modified analogs:

Last in the series, site F modified compounds were obtained by coupling different carboxylic acid analogs with compound 8a to obtain several sulfonamide analogs. This included increasing bis-alkyl chain length on sulfonamide analogs 54a (N-ethyl, N-propyl), 54b (N,N-dipropyl), and 54c (N,N-dibutyl) (Scheme 7). Up to a propyl chain length substituent was tolerated while dibutyl substituted compound 54c lost activity (Table 1). Thus, bulky steric groups may not be tolerated. Replacing one alkyl chain with a phenyl group afforded 54d while extending the carbocyclic ring by one atom yielded N-cyclohexyl sulfonamide analog 54e. Both these molecules were inactive indicating that the aryl substitution or mono-alkyl substitution on sulfonamide were not tolerated (Table 1). The preliminary SAR suggested that all compounds bearing sulfonamide linked to piperazine were inactive (Fig. 1), but this set of compounds only included ethyl carbamate linked to 4-nitrogen of the piperazine ring, while the piperazine or its N-amide and N-alkyl analogs were not explored. Thus, en route towards the syntheses of substituted piperazine analogs, we made N-Boc protected piperazine compound 54f. The N-Boc was removed to obtain compound 54i, followed by acetylation to obtain 54j or alkylation to obtain N-propyl analog 54k (Scheme 7). All the piperazine bearing molecules were found to be inactive, prompting us to focus on the substituted piperidine analogs (Table 1). Thus, we synthesized 4-ethoxy and 4-propyl substituted piperidine analogs 54g and 54h, respectively, as shown in Scheme 7. Both these 4-substituted piperidine analogs were found to be very potent (Table 1).

Scheme 7. Syntheses of sites F modified compounds.

Reagents and conditions: i. For X = H: Et₃N, CH₂Cl₂. For X = -CH₃: (a) Et₃N, CH₂Cl₂, (b) LiOH, MeOH, THF, H₂O; ii. 8a, HATU, Et₃N, DMF, 45 °C; iii. 4N HCl/dioxane; iv. CH₃COCl, Et₃N, DMF; v. C₃H₇I, K₂CO₃, DMF.

Hybrid compounds:

All the SAR studies so far led us to identify three compounds, namely 12d, 18q, and 54h each from site A, B, and F modified, respectively; that were most active in NF-κB inducing assays (Table 1, Supporting Information Fig. S3). These compounds bear structural features that made them more potent than compound 1 and we wondered if synergistic activities could be obtained by combining these chemical features to obtain hybrid compounds. Combining the structural feature of 2-bromo substitution on the 4-phenylthiazole as in 12d (site A modified compound) with the 4-propylpiperidine feature as in 54h (site F modified compound) yielded compound 59 as shown in Scheme 8. Similarly, combining the 5-pentynyl substitution as in 18q (Site B modified compound) and 4-propylpiperidine feature as in 54h provided compound 61. Similarly, combining features of compound 12d and 18q provided Site A and B modified hybrid compound 60. Inclusion of all the structural features of the above-mentioned compounds led us to compound 61 (Scheme 8). Bioactivity analysis of these hybrid compounds showed that while compounds 55 and 60 retained NF-κB activity, they did not show any synergistic increase in activity. In contrast, compounds 57 and 61, both of which had the 4-propylpiperidine substituent as Site F, led to significant loss of activity. Ideally, combining the chemical attributes such as substituents necessary for activity in potent compounds leads to enhancement of activity, however, in this case, combining the structural features of potent compounds to obtain hybrid compounds did not yield any synergistic effects. This could possibly be due to overall enhancement of the hydrophobicity or the size of these hybrid compounds.

Scheme 8. Syntheses of hybrid analogs.

Reagents and conditions: i. (a) HATU, Et₃N, DMF, 45 °C; ii. pentyne, Pd(PPh₃)₄, Cul, DMF; iii 53h, HATU, Et₃N, DMF; iv. N-iodosuccinimide, CH₂Cl₂; v. for compound 60: 4, HATU, Et₃N, DMF, 45 °C and for compound 61: 53h, HATU, Et₃N, DMF, 45 °C.

Cellular toxicity assessment:

All these compounds were evaluated for toxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. MTT data analysis showed that most of the compounds did not exhibit any significant toxicity with %viability >80% of vehicle control values. Some of the compounds enhanced cell growth as seen by %viability over 100% (Table 1). The few exceptions included a site B modified imidazole group (18a), a substituent swap analog (51) and a site F modified 4-propylpiperazine bearing compound (54k) that showed lower than 80% viability despite the compound being inactive in NF-κB assays.

Dose response NF-κB activity profile:

The bioactivity data (Table 1) and the SAR studies pointed us to the most potent compounds on sites A, B and F as 12d, 18q and 54h, respectively (Supporting Information Fig. S3). These compounds selected based on the NF-κB SEAP assay were further characterized by dose responses in NF-κB induction in combination with LPS as these compounds prolonged NF-κB activation in the HTS (Fig. 3). The dose response curves more finely differentiated the potencies in terms of EC₅₀ values in the presence of LPS (10 ng/mL). Compound 12d showed a rapid fall in activity at 2.5 μM (EC₅₀ = 4.5 μM) compared to compound 1 with EC₅₀ of 2.3 μM. Compounds 18q (EC₅₀ = 1.7 μM) and 54h (EC₅₀ = 0.9 μM) were more potent than compound 1.

Figure 3.

Dose response curves for selected active analogs with LPS. Compounds 1, 12d, 18q, and 54h were evaluated for enhancement of NF-κB signaling at graded concentrations. Site A modified compound 12d was less potent than compound 1 while sites B and F modified compounds 18q and 54h were more potent. The activity of LPS alone is shown as a grey bar. Data are presented as mean ± SEM for experiments performed in triplicates in two independent experiments. The NF-κB activation was measured as SEAP induced in the culture supernatant.

Cytokine and chemokine induction:

Activation of NF-κB is usually characterized by production of cytokines and chemokines including IL-6, IL-8, IL-12 and TNF-α. Because NF-κB controls multiple intracellular functions, including cytokine and chemokine expression, we were interested to see if the potent compounds enhanced production of a representative cytokine. THP-1 cells (wild type) were treated with compounds with and without LPS and NF-κB downstream chemokine IL-8 was measured by ELISA. The selected compounds (5 μM concentration) significantly enhanced the IL-8 production when co-incubated with LPS (1 ng/mL) compared to LPS alone. When cells were treated with compound alone, the treated cells released significantly higher levels of IL-8 in comparison to the vehicle control (Fig. 4).

Figure 4.

IL-8 release by selected active analogs with and without LPS in THP-1 cells. Compounds 1, 12d, 18q and 54h (5 μM) not only induced IL-8 but significantly enhanced its induction in the presence of LPS (1 ng/mL, shaded boxes) and alone as well (unshaded boxes). Compounds 1 and 54h also induced significantly higher cytokines compared to that of 12d and 18q. **p<0.001 and *p<0.01 compared to Veh or Veh+LPS by one-way ANOVA analysis and Bonferroni’s post hoc testing.

Since the aim of this project is to verify the adjuvanticity of compounds in an animal model such as mice, we chose to first verify if these compounds induced cytokine secretion by primary murine derived bone marrow dendritic cells (BMDCs) similar to that observed in the human THP-1 cell line. Murine BMDCs treated with compound alone did not release detectable amounts of IL-12, but when BMDCs were treated with compounds in the presence of LPS, a significant enhancement in IL-12 secretion in the culture supernatant was observed compared to IL-12 secretion induced by LPS alone (Supporting Information Fig. S4). The cytokine induction profile for these compounds in murine BMDCs was similar to IL-8 induction in human THP-1 cells illustrating their activity in both human and mice cells as well as primary immune cells.

In vivo adjuvanticity:

Encouraged by the potency of compounds in both human and murine cells, we were interested to verify the potency as co-adjuvants with MPLA (FDA approved TLR-4 agonistic adjuvant), for in vivo evaluation. Immunization experiments in mice (5 mice/group) were performed to evaluate the co-adjuvanticity of these selected lead compounds (50 nmol/mice) with low dose of MPLA (10 ng/mice) using ovalbumin (OVA, 20 μg/mice) as a model antigen. Examination of OVA-specific IgG antibodies showed that co-immunization of MPLA with compounds 18q and 54h induced statistically significant increases in antigen-specific antibody titers when compared to mice immunized with MPLA alone (Fig. 5, Left). There was also no demonstrable systemic toxicity, as indicated by behavior change or weight loss. These results showed that selected potent compounds that prolonged NF-κB stimulation enhanced the adjuvanticity of MPLA. Although adjuvanticity is a complex biological response involving different immune cell types, tissue distribution, antigen presentation and antibody maturation, we wondered if there was any correlation between NF-κB inducing activity and total IgG responses to immunization for these 4 potent compounds. We plotted the mean anti-OVA IgG titers obtained post immunization against potency (EC₅₀s from Fig. 3) of these compounds in the THP-1 NF-κB SEAP assay (Fig. 5, Right). It is notable that there was good correlation (P<0.0007, R² = 0.9986; Pearson two-tailed correlation) between the mean antibody titers and compound potency in the THP-1 reporter cells. The results suggested that dose response potency measurements (EC₅₀ calculation) in vitro may be a useful tool to predict adjuvantic potency in vivo for these series of compounds.

Figure 5.

Co-adjuvanticity of potent analogs with MPLA. Left: mice (n=5 per group) were immunized on day 0 and day 21 with antigen (OVA, 20 μg/mice), MPLA (10 ng/mice) and compound 1, 12d, 18q or 54h (50 nmol/mice). The immunized mice were bled on day 28 and OVA-specific IgG titers were measured using ELISA. Left: Box and whisker plot showing statistically significant enhancement of antibody titers for compounds 18q and 54h when co-adjuvanted with MPLA compared to MPLA alone. **p<0.05 compared to MPLA group using Bartlett’s one-way ANOVA followed by Dunnett’s post hoc testing. Right: Pearson two-tailed correlation ((P<0.0007, R² = 0.9986) of mean (positive standard deviation) of OVA-specific IgG titers and EC₅₀ values obtained for NF-κB SEAP assay in THP-1 cells with LPS.

Affinity probe:

The further drug development of HTS hits like compound 1 which are identified through cell-based phenotypic assays and are involved in cell signaling pathways can likely proceed with additional knowledge of the target receptor or the compound’s mechanism of action.³² Thus, another aspect of SAR studies is to identify positions on the scaffold that can tolerate the introduction of small functional groups such as aryl azide or diazirine to make photoreactive probes.^{29, 33-37} Such chemical probes would then be useful tools for future mechanistic and functional receptor studies. Based on the data here and our experience with design and synthesis of photoreactive probes, we decided to introduce azide functionality at site A by displacing bromo to obtain 3-azido and 2-azido analogs 63 and 66, respectively as shown in Scheme 9. While 2-azido compound 66 was inactive, the 3-azido compound 63 retained activity, thus allowing us to utilize site A for introducing a photoreactive aryl azide functional group.

Scheme 9. Syntheses of aryl azide and alkyne bearing analogs.

Reagents and conditions: i. Cul, 1,2-Dimethylethylenediamine, NaN₃, DMF, microwave 100 °C; ii. 4, HATU, Et₃N, DMF; iii. (a) Br₂, CHCl₃, (b) thiourea, EtOH, 100 °C; iv. Et₃N, CH₂Cl₂; v. 8a, HATU, Et₃N, DMF.

Next, in order to introduce a reactive tag such as an alkyne for pull down approaches, we decided to probe site F. A series of propargyl bearing compounds such as N-propargyl 68a, N-ethyl,N-propargyl 68b and N-propyl,N-propargyl 68c were synthesized as shown in Scheme 9. While the fully reduced compounds synthesized earlier including analogs 54a and 54b (Scheme 7) were active (Table 1), the corresponding alkyne bearing compounds (68a and 68b) lost their activity. We next utilized the position 4 of the piperidine ring to introduce the ethyne group to obtain compound 68d. Bioactivity data showed that 68d was modestly active which allowed us to proceed towards making the photoaffinity probe by combining the active aryl azide attribute of compound 63 and alkyne attribute of compound 68d. This was done by HATU coupling of compounds 62 and 67d to obtain photoaffinity probe 69 as shown in Scheme 10. As a test of potency of the photoaffinity probe 69, we compared it against compound 1 for NF-kB induction in presence of LPS. A perfect overlapping dose-response curve (Fig. 6) confirmed the activity of the photoaffinity probe which will be utilized in future for target identification studies.

Scheme 10. Synthesis of photoaffinity probe.

Reagents and conditions: i. HATU, Et₃N, DMF.

Figure 6.

Potency of analogs towards design of photoaffinity probe. A) Potency of photoreactive aromatic azide and alkyne bearing analogs compared to compound 1. Azide bearing compound 63 and alkyne bearing compound 68d were identified to be active in inducing NF-κB induction in the presence of LPS. B) Comparison of dose-dependent NF-κB enhancing activity in presence of LPS of compound 1 and photoaffinity probe 69 showing identical potency.

Conclusion:

Compounds bearing sulfamoyl benzamidothiazole functionalities were identified through HTS that enhance NF-κB signaling from a primary TLR-4 stimulus. Systematic SAR studies involving six different sites of modifications on the scaffold led to identification of more potent compounds than the original hit compound 1. These compounds not only enhanced the NF-κB response but also augmented the induction of immunostimulatory IL-8 in THP-1 cells and IL-12 in mouse BMDCs. Overall, these compounds were found to be safe in both in vitro and in vivo assays and did not induce systemic inflammatory responses. Vaccine co-adjuvanticity experiments in mice showed that these compounds enhanced antigen-specific antibody responses with the adjuvant MPLA compared to MPLA alone. These studies indicate that future evaluation of these potent compounds in infectious disease and vaccine models is warranted. Additionally, the SAR studies presented here also helped synthesize a photoaffinity probe which will further help in target identification and mechanisms of action studies.

Experimental Section:

Chemistry

Materials.

Reagents were purchased as at least reagent grade from commercial vendors unless otherwise specified and used without further purification. Solvents were purchased from Fischer Scientific (Pittsburgh, PA) and were either used as purchased or redistilled with an appropriate drying agent. Compounds used for SAR studies were synthesized according to methods described below and all the compounds were identified to be at least 95% pure using HPLC.

Instrumentation.

Analytical TLC was performed using precoated TLC silica gel 60 F₂₅₄ aluminum sheets purchased from EMD (Gibbstown, NJ) and visualized using UV light. Flash chromatography was carried out using a Biotage Isolera One (Charlotte, NC) system for normal phase column chromatography or Teledyne ISCO ACCQPrep HP150 for C18-reverse phase column chromatography using the specified solvent. Microwave reactions were performed using Biotage Initiator+ (Charlotte, NC). Reaction monitoring and purity analysis were done using an Agilent 1260 LC/6420 Triple Quad mass spectrometer (Santa Clara, CA) with Onyx Monolithic C18 (Phenomenex, Torrance, CA) column. Purity of all final compounds was above 95% (also see LC-MS spectra in Supporting Information for all final compounds). All final compounds were analyzed by high resolution MS (HRMS) using an Agilent 6230 ESI-TOFMS (Santa Clara, CA). ¹H and ¹³C NMR spectra were obtained on a Varian 500 with XSens probe (Varian, Inc., Palo Alto, CA). The chemical shifts are expressed in parts per million (ppm) using suitable deuterated NMR solvents.

Compound 1 and site A modified compounds 12b-f, p-s were synthesized using general procedures used for synthesis of compound 1 as described below.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (1).

Compound 1 was synthesized using two advanced intermediates 4 and 8a which were synthesized as shown below.

Synthesis of compound 4: 4-(piperidin-1-ylsulfonyl)benzoic acid.

To a solution of 4-(chlorosulfonyl)phenyl acetate (2, 500 mg, 2.14 mmol) in anhydrous dichloromethane were added triethylamine (432 mg, 4.28 mmol) and piperidine (3, 200 mg, 2.35 mmol). The reaction mixture was stirred for an hour followed by removal of the solvent to obtain the crude sulfonamide intermediate methyl 4-(piperidin-1-ylsulfonyl)benzoate, which was suspended in water and filtered to remove the water soluble impurities, followed by dissolving the residue in 3:1 mixture of MeOH/THF. Lithium hydroxide (LiOH, 268 mg, 6.4 mmol) dissolved in 0.5 mL of water was added to this solution and stirred for another 2h followed by removal of the solvent under vacuum. The residue was then dissolved in water and the solution was acidified using 3M HCl to precipitate the product which was purified by filtration to obtain compound 4 as white solid (511 mg, yield = 84%).

Synthesis of compound 8a:

To a solution of 1-(2,5-dimethylphenyl)ethan-1-one (6a, 2g, 13.5 mmol) in chloroform was added liquid bromine (2.38 g, 14.9 mmol). The reaction was stirred for an hour, followed by removal of the solvent to obtain crude intermediate 7a which was dissolved in anhydrous EtOH and thiourea was added. The reaction was then heated in microwave at 100 °C for 30 min, followed by removal of the solvent to obtain the residue which was purified using silica gel column chromatography to obtain compound 8a as tan solid (1.8 g, yield = 65%).

The “HATU coupling” reaction for forming the amide bond will be utilized for syntheses of several other compounds which will follow the protocol shown here for synthesis of compound 1:

To a solution of compound 4 (50 mg, 0.19 mmol) in anhydrous DMF were added, 8a (42 mg, 0.21 mmol), HATU (78 mg, 0.21 mmol), and triethylamine (28 mg, 0.28 mmol) and the reaction was heated at 45 °C for 16h. The solvent was then removed and the residue was suspended in aqueous sodium bicarbonate solution. The precipitate was filtered to obtain crude product which was purified using silica gel column chromatography to yield compound 1 (65 mg, yield = 77%) as white solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.14 (br. s., 1H), 7.80 (d, J = 8.31 Hz, 2H), 7.63 (d, J = 8.56 Hz, 2H), 7.14 (s, 1H), 7.00 (s, 1H), 6.98 (d, J = 7.58 Hz, 1H), 6.87 - 6.93 (m, 1H), 2.93 - 3.01 (m, 3H), 2.29 (s, 3H), 2.24 (s, 2H), 1.61 - 1.68 (m, 4H), 1.39 - 1.46 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.0, 158.4, 150.0, 139.7, 135.4, 135.3, 133.3, 132.5, 130.9, 129.9, 129.0, 128.0, 127.6, 111.4, 46.8, 25.1, 23.4, 20.8, 20.3. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H]⁺ calculated 456.1410, found 456.1408.

Compounds 12b-f, 12p were synthesized using the procedure similar to compound 1 and corresponding intermediates 8b-f, 8p were synthesized similarly as compound 8a.

4-(piperidin-1-ylsulfonyl)-N-(4-(o-tolyl)thiazol-2-yl)benzamide (12b).

1-(o-tolyl)ethan-1-one (6b, 200 mg, 1.49 mmol) was converted to compound 8b which was followed by ‘HATU coupling’ reaction with compound 4 (25 mg, 0.093 mmol) to obtain 12b (40 mg, yield = 97% for the final step) as off-white solid. Ή ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.77 (br. s., 1H), 7.82 (d, J = 8.07 Hz, 2H), 7.68 (d, J = 8.07 Hz, 2H), 7.34 (d, J = 6.60 Hz, 1H), 7.07 - 7.21 (m, 3H), 7.01 (s, 1H), 2.98 (d, J = 4.40 Hz, 4H), 2.35 (s, 3H), 1.63 - 1.70 (m, 4H), 1.43 (d, J = 5.14 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.0, 158.2, 150.0, 139.9, 135.7, 135.5, 133.6, 130.9, 129.3, 128.3, 128.0, 127.8, 126.0, 111.5, 46.9, 25.1, 23.4, 20.8. HRMS for C₂₂H₂₄N₃O₃S₂ [M + H]⁺ calculated 442.1254, found 442.1251.

4-(piperidin-1-ylsulfonyl)-N-(4-(m-tolyl)thiazol-2-yl)benzamide (12c).

1-(m-tolyl)ethan-1-one (6c, 200 mg, 1.49 mmol) was converted to compound 8c which was followed by ‘HATU coupling’ reaction with compound 4 (25 mg, 0.093 mmol) to obtain 12c (18 mg, yield = 44% for the final step) as tan solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.10 (br. s., 1H), 7.92 - 7.95 (m, 2H), 7.73 (d, J = 8.31 Hz, 2H), 7.49 - 7.54 (m, 2H), 7.20 - 7.24 (m, 2H), 7.08 (d, J = 7.58 Hz, 1H), 2.95 - 3.00 (m, 4H), 2.36 (s, 3H), 1.61 - 1.67 (m, 4H), 1.40 - 1.49 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.8, 158.5, 150.4, 140.1, 138.5, 135.5, 133.7, 129.0, 128.0, 127.9, 126.7, 123.2, 108.4, 46.9, 25.1, 23.4, 21.5. HRMS for C22H24N3O3S2 [M + H]⁺ calculated 442.1254, found 442.1250.

N-(4-(2-bromo-5-methylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12d).

The procedure to synthesize compound 6d needed for synthesis of 12d is shown below. To a solution of 2-bromo-5-methylbenzoic acid (5d, 200 mg, 0.93 mmol) in anhydrous DMF were added, HATU (388 mg, 1.02 mmol), triethylamine (235 mg, 2.33 mmol) and N,O-dimethyl hydroxylamine hydrochloride (100 mg, 1.02 mmol). The reaction was stirred for an hour followed by removal of the solvent under vacuum to obtain the residue, which was dissolved in EtOAc, washed with aqueous sodium bicarbonate solution and dried over sodium sulfate to obtain the Weinreb amide intermediate. This was dissolved in anhydrous THF followed by the addition of methyl magnesium bromide (1 mL) under anhydrous conditions on ice and the reaction was then heated at 45 °C for 16h. The reaction was quenched with 3M HCl solution, followed by extraction of compound in EtOAc. The EtOAc fraction was washed with water, dried over sodium sulfate and concentrated under vacuum to obtain the residue which was purified using column chromatography to obtain compound 6d as clear oil. Compound 6d (200 mg, 1.49 mmol) was converted to compound 8d which was followed by ‘HATU coupling’ reaction with compound 4 (50 mg, 0.19 mmol) to obtain 12d (37 mg, yield = 48% for the final step) as white solid. ¹H NMR (500 MHz, CHLOROFORM-d) d 11.59 (br. s., 1H), 7.92 - 7.96 (m, 2H), 7.75 (d, J = 8.56 Hz, 2H), 7.36 - 7.41 (m, 2H), 7.30 (d, J = 1.96 Hz, 1H), 6.85 - 6.90 (m, 1H), 2.97 - 3.04 (m, 4H), 2.27 (s, 3H), 1.65 - 1.69 (m, 4H), 1.40 - 1.48 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.0, 159.7, 145.3, 140.8, 137.8, 134.5, 133.5, 132.0, 131.3, 128.7, 118.7, 113.0, 46.9, 25.1, 23.4, 20.8. HRMS for C₂₂H₂₃BrN₃O₃S₂ [M + H]⁺ calculated 520.0359, found 520.0354.

N-(4-(5-bromo-2-methylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12e).

Compound 12e was synthesized using the same procedure as 12d starting with 5-bromo-2-methylbenzoic acid (5e, 200 mg, 0.93 mmol) to obtain 6e (162 mg, yield = 82%) as clear oil. Compound 6e (160 mg, 0.75 mmol) was converted to compound 8e which was followed by ‘HATU coupling’ reaction with compound 4 (110 mg, 0.41 mmol) to obtain 12e (79 mg, yield = 37% for the final step) as white solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.23 (br. s., 1H), 7.93 - 7.98 (m, 2H), 7.74 - 7.78 (m, 2H), 7.61 (d, J = 2.20 Hz, 1H), 7.25 (s, 1H), 7.02 - 7.07 (m, 2H), 2.96 - 3.04 (m, 4H), 2.37 (s, 3H), 1.61 - 1.65 (m, 4H), 1.40 - 1.47 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 158.0, 148.2, 140.3, 135.6, 135.4, 134.6, 132.7, 132.0, 131.0, 128.0, 127.9, 119.4, 112.4, 46.9, 25.1, 23.4, 20.7. HRMS for C₂₂H₂₃BrN₃O₃S₂ [M + H⁺] calculated 520.0359, found 520.0353.

N-(4-(2,5-dimethylbenzyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12f).

Compound 6f (200 mg, 1.24 mmol) was converted to compound 7f which was followed by ‘HATU coupling’ reaction with compound 4 (16 mg, 0.07 mmol) to obtain 12f (23 mg, yield = 70% for the final step) as white solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.36 (d, J = 8.31 Hz, 2H), 7.95 (d, J = 8.31 Hz, 2H), 7.03 - 7.14 (m, 3H), 6.40 (s, 1H), 4.08 (s, 2H), 3.06 (t, J = 5.14 Hz, 4H), 2.33 (s, 3H), 2.25 (s, 3H), 1.67 (quin, J = 5.60 Hz, 4H), 1.43 - 1.48 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.2, 161.4, 145.1, 141.2, 136.1, 134.0, 133.9, 133.3, 130.9, 130.6, 129.1, 128.4, 128.2, 109.4, 46.9, 33.1, 25.1, 23.4, 20.9, 18.9. HRMS for C₂₄H₂₈N₃O₃S₂ [M + H]⁺ calculated 470.1567, found 470.1562.

Site A modified compounds 12g-k were synthesized starting with compounds 8g-k (purchased from Enamine LLC) respectively using the ‘HATU coupling’ reaction procedure described above for synthesis of compound 1.

N-(4-methylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12g).

Compound 12g was synthesized using 4-methylthiazol-2-amine (8g, 8.5 mg, 0.074 mmol) and compound 4 (20 mg, 0.074 mmol) as light yellow solid (22 mg, yield = 82%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.37 (br. s., 1H), 8.03 - 8.07 (m, 2H), 7.87 (dd, J = 1.10, 8.44 Hz, 2H), 6.62 (t, J = 0.98 Hz, 1H), 3.03 (t, J = 5.14 Hz, 4H), 2.09 (d, J = 0.98 Hz, 3H), 1.66 (td, J = 5.59, 11.07 Hz, 4H), 1.41 - 1.48 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.8, 158.5, 146.8, 140.4, 136.1, 128.3, 128.1, 109.0, 46.9, 25.1, 23.4, 16.6. HRMS for C₁₆H₂₀N₃O₃S₂ [M + H]⁺ calculated 366.0941, found 366.0937.

N-(4-ethylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12h).

Compound 12h was synthesized using 4-ethylthiazol-2-amine (8h, 9.5 mg, 0.074 mmol) and compound 4 (20 mg, 0.074 mmol) as off-white solid (16 mg, yield = 57%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.76 (br. s., 1H), 8.06 (d, J = 8.07 Hz, 2H), 7.87 (d, J = 8.07 Hz, 2H), 6.63 (d, J = 0.73 Hz, 1H), 3.02 (t, J = 5.38 Hz, 4H), 2.52 (q, J = 7.58 Hz, 2H), 1.66 (td, J = 5.72, 11.31 Hz, 4H), 1.44 (quin, J = 5.81 Hz, 2H), 1.20 (t, J = 7.46 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 158.1, 153.1, 140.3, 136.0, 128.2, 128.1, 107.6, 46.9, 25.1, 24.4, 23.4, 13.0. HRMS for C₁₇H₂₂N₃O₃S₂ [M + H]⁺ calculated 380.1097, found 380.1094.

4-(piperidin-1-ylsulfonyl)-N-(4-propylthiazol-2-yl)benzamide (12i).

Compound 12i was synthesized using 4-propylthiazol-2-amine hydrochloride (8i, 13.3 mg, 0.074 mmol) and compound 4 (20 mg, 0.074 mmol) as white solid (27 mg, yield = 93%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.52 (br. s., 1H), 8.07 (d, J = 8.32 Hz, 2H), 7.88 (d, J = 8.31 Hz, 2H), 6.63 (s, 1H), 3.03 (t, J = 4.89 Hz, 4H), 2.50 (t, J = 7.58 Hz, 2H), 1.60 - 1.69 (m, 6H), 1.38 - 1.53 (m, 2H), 0.92 (t, J = 7.34 Hz, 4H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.5, 158.0, 151.6, 140.4, 136.0, 128.2, 128.1, 108.3, 46.9, 33.2, 25.1, 23.4, 22.1, 13.7. HRMS for C₁₈H₂₄N₃O₃S₂ [M + H]⁺ calculated 394.1254, found 394.1251.

N-(4-neopentylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12j).

Compound 12j was synthesized using 4-neopentylthiazol-2-amine (8j, 12.6 mg, 0.074 mmol) and compound 4 (20 mg, 0.074 mmol) as off-white solid (26 mg, yield = 84%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.80 (br. s., 1H), 8.04 - 8.09 (m, 2H), 7.85 - 7.90 (m, 2H), 6.62 (s, 1H), 3.00 - 3.05 (m, 4H), 2.35 (s, 2H), 1.66 (td, J = 5.78, 11.19 Hz, 4H), 1.41 - 1.48 (m, 2H), 0.87 (s, 9H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.5, 157.4, 149.5, 140.3, 136.0, 128.2, 128.1, 110.5, 46.9, 44.9, 31.5, 29.4, 25.1, 23.4. HRMS for C₂₀H₂₈N₃O₃S₂ [M + H]⁺ calculated 422.1567, found 422.1565.

N-(4-(tert-butyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12k).

Compound 12k was synthesized using 4-(tert-butyl)thiazol-2-amine hydrochloride (8k, 14.4 mg, 0.074 mmol) and compound 4 (20 mg, 0.074 mmol) as off-white solid (26 mg, yield = 86%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 9.63 (br. s., 1H), 8.10 (dd, J = 1.22, 8.31 Hz, 2H), 7.88 - 7.93 (m, 2H), 6.61 - 6.65 (m, 1H), 3.03 (t, J = 5.26 Hz, 4H), 1.66 (quin, J = 5.56 Hz, 4H), 1.45 (d, J = 5.14 Hz, 2H), 1.32 (s, 9H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.0, 161.0, 156.9, 140.3, 135.9, 128.2, 128.0, 105.8, 46.9, 34.4, 29.8, 25.1, 23.4. HRMS for C₁₉H₂₆N₃O₃S₂ [M + H]⁺ calculated 408.1410, found 408.1407.

tert-Butyl (4-bromothiazol-2-yl)carbamate) (10).

This reaction was performed by halogen dance reaction as reported in the literature.³⁰ To a cooled solution of diisopropylamine (1.12 g, 11.1 mmol) in anhydrous THF (20 mL) was slowly added 1.6M n-butyllithium solution in hexanes (690 mg, 10.8 mmol). The reaction was allowed to cool again for 5 min followed by dropwise addition of a solution of tert-butyl (5-bromothiazol-2-yl)carbamate (9, 1 g, 3.6 mmol) in anhydrous THF (20 mL). The reaction was stirred for 30 min, followed by quenching the reaction with addition of 2 mL of water. EtOAc was added and the organic layer was separated. The EtOAc fraction was then washed with saturated NH₄Cl solution, brine, dried over sodium sulfate and concentrated under vacuum to obtain residue which was purified using silica gel column chromatography to obtain compounds 10 as tan solid (635 mg, yield = 63.5%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.39 (br. s., 1H), 6.81 (s, 1H), 1.55 (s, 9H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 160.1, 151.8, 120.6, 110.4, 83.3, 28.1. MS for C₈H₁₁BrN₂O₂S [M + Na⁺] calculated 301.0, found 300.8.

N-(4-(2,3-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12l).

The Suzuki coupling reaction was first performed to synthesize 11l as described below. To a microwave reaction vial, tetrakis(triphenylphosphine) palladium(0) (103 mg, 0.09 mmol) and 2,4-dimethylphenylboronic acid (32 mg, 0.22 mmol) were combined and the vial was sealed and evacuated. Compound 10 (50 mg, 0.18 mmol) dissolved in anhydrous DMF was then added to the vial. Separately, sodium carbonate (57 mg, 0.54 mmol) dissolved in water was also added and the vial was filled with argon gas. The reaction mixture was then irradiated in the microwave at 100 °C for 30min. After concentration, the residue was dissolved in EtOAc and washed with brine, dried over sodium sulfate, and then concentrated under vacuum. The resulting residue was then purified by silica gel column chromatography to obtain compound 11l (22 mg, yield = 40%). The N-Boc was then removed by stirring compound 11l (22 mg, 0.072 mmol) in 3 mL of 4N HCl solution in dioxane for 1h, followed by removal of solvent to obtain residue which was purified using column chromatography to obtain compound 8l (15 mg, yield = 87%). The ‘HATU coupling’ reaction was then performed using 4 (20 mg, 0.074 mmol), 8l (15.5 mg, 0.076 mmol), HATU (34 mg, 0.089 mmol), and DIPEA (22 mg, 0.17 mmol) to obtain compound 12l as white solid (7 mg, yield = 21%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.15 (br. s., 1H), 7.82 (d, J = 8.31 Hz, 2H), 7.65 (d, J = 8.31 Hz, 2H), 7.05 - 7.14 (m, 1H), 6.96 - 7.03 (m, 2H), 6.94 (s, 1H), 2.97 (t, J = 4.77 Hz, 4H), 2.22 (s, 3H), 2.16 (s, 3H), 1.60 - 1.66 (m, 4H), 1.38 - 1.47 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.1, 158.6, 150.5, 139.7, 137.6, 135.4, 134.3, 133.9, 129.9, 128.0, 127.6, 127.4, 125.6, 111.5, 46.9, 25.1, 23.4, 20.5, 16.9. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H]⁺ calculated 456.1410, found 456.1404.

Compounds 12m-o were synthesized using the same procedure as for compound 12l.

N-(4-(2,4-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12m).

The Suzuki coupling reaction was performed using tetrakis(triphenylphosphine) palladium(0) (103 mg, 0.09 mmol), 2,4-dimethylphenylboronic acid (32 mg, 0.22 mmol), 10 (50 mg, 0.18 mmol) and sodium carbonate (57 mg, 0.54 mmol) to obtain compound 11m (28 mg, yield = 51%). N-Boc removal on 11m using 4N HCl solution in dioxane provided 8m (18 mg, yield = 96%). The ‘HATU coupling’ reaction was then performed using 4 (26 mg, 0.1 mmol), 8m (18 mg, 0.088 mmol), HATU (37 mg, 0.097 mmol), and DIPEA (25 mg, 0.194 mmol) to obtain compound 12m as off-white solid (10 mg, yield = 23%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.07 (br. s., 1H), 7.80 (d, J = 8.31 Hz, 2H), 7.66 (d, J = 8.56 Hz, 2H), 7.20 (d, J = 7.58 Hz, 1H), 6.96 (s, 1H), 6.85 - 6.92 (m, 2H), 2.93 - 3.03 (m, 4H), 2.30 (s, 3H), 2.26 (s, 3H), 1.61 - 1.66 (m, 4H), 1.37 - 1.47 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.1, 158.4, 150.0, 139.7, 138.0, 135.5, 135.4, 131.7, 130.7, 129.3, 128.0, 127.6, 126.7, 111.0, 46.9, 25.1, 23.4, 21.0, 20.7. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H]⁺ calculated 456.1410, found 456.1405.

N-(4-(3,4-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12n).

The Suzuki coupling reaction was performed using tetrakis(triphenylphosphine) palladium(0) (103 mg, 0.09 mmol), 2,4-dimethylphenylboronic acid (32 mg, 0.22 mmol), 10 (50 mg, 0.18 mmol) and sodium carbonate (57 mg, 0.54 mmol) to obtain compound 11n (9 mg, yield = 17%). N-Boc removal on 11n 4N HCl solution in dioxane provided 8n (6 mg, quantitative yield). The ‘HATU coupling’ reaction was then performed using 4 (8.7 mg, 0.032 mmol), 8n (6 mg, 0.029 mmol), HATU (12 mg, 0.032 mmol), and DIPEA (8.4 mg, 0.065 mmol) to obtain compound 12n as off-white solid (4.2 mg, yield = 32%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.03 (br. s., 1H), 7.96 (d, J = 8.07 Hz, 2H), 7.74 (d, J = 7.83 Hz, 2H), 7.39 - 7.49 (m, 2H), 7.13 - 7.19 (m, 1H), 7.08 (d, J = 7.83 Hz, 1H), 2.98 (d, J = 3.91 Hz, 4H), 2.20 - 2.29 (m, 6H), 1.65 (br. s., 4H), 1.43 (br. s.,2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 158.3, 150.5, 140.1, 137.0, 136.9, 135.6, 131.5, 130.0, 128.0, 127.9, 127.3, 123.5, 107.7, 46.9, 25.1, 23.4, 19.9, 19.6. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H]⁺ calculated 456.1410, found 456.1403.

N-(4-(3,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12o).

The Suzuki coupling reaction was performed using tetrakis(triphenylphosphine) palladium(0) (103 mg, 0.09 mmol), 2,4-dimethylphenylboronic acid (32 mg, 0.22 mmol), 10 (50 mg, 0.18 mmol) and sodium carbonate (57 mg, 0.54 mmol) to obtain compound 11o (16 mg, yield = 29%). N-Boc removal on 11o 4N HCl solution in dioxane provided 8o (12.4 mg, quantitative yield). The ‘HATU coupling’ reaction was then performed using 4 (11.5 mg, 0.043 mmol), 8o (12.4 mg, 0.052 mmol), HATU (34 mg, 0.09 mmol), and DIPEA (12.6 mg, 0.098 mmol) to obtain compound 12o as off-white solid (16.5 mg, yield = 84%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.33 (d, J = 8.31 Hz, 2H), 7.94 (d, J = 8.31 Hz, 2H), 7.39 (s, 2H), 7.16 (s, 1H), 7.09 (s, 1H), 3.06 (t, J = 5.38 Hz, 4H), 2.39 (s, 6H), 1.67 (quin, J = 5.69 Hz, 4H), 1.44 - 1.48 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.5, 161.6, 145.9, 141.1, 139.0, 134.2, 131.5, 129.7, 129.1, 128.2, 124.4, 107.8, 46.9, 25.1, 23.4, 21.3. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H]⁺ calculated 456.1410, found 456.1403.

N-(4-(2,6-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12p).

1-(2,6-dimethylphenyl)ethan-1-one (6p, 100 mg, 0.67 mmol) was converted to compound 8p which was followed by ‘HATU coupling’ reaction with compound 4 (20 mg, 0.074 mmol) to obtain 12p (17 mg, yield = 51% for the final step) as white solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.65 (br. s., 1H), 7.88 (d, J = 8.31 Hz, 2H), 7.77 (d, J = 8.31 Hz, 2H), 6.98 - 7.02 (m, 1H), 6.90 (d, J = 7.58 Hz, 2H), 6.83 (s, 1H), 3.02 (t, J = 5.26 Hz, 4H), 2.00 (s, 6H), 1.63 - 1.68 (m, 4H), 1.41 - 1.48 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.8, 158.5, 148.4, 140.0, 136.9, 135.4, 133.7, 128.3, 128.0, 127.8, 127.5, 112.0, 46.9, 25.1, 23.4, 20.4. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H]⁺ calculated 456.1410, found 456.1404.

N-(4-(naphthalen-1-yl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12q).

Compound 12q was synthesized using ‘HATU coupling’ reaction using 4-(naphthalen-1-yl)thiazol-2-amine (8q, 25 mg, 0.11 mmol) and compound 4 (29 mg, 0.11 mmol) as light-yellow solid (33 mg, yield = 63%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.05 (br. s., 1H), 8.17 (d, J = 8.56 Hz, 1H), 7.78 (d, J = 8.07 Hz, 1H), 7.67 - 7.75 (m, 3H), 7.50 - 7.59 (m, 3H), 7.48 (t, J = 7.34 Hz, 1H), 7.42 (t, J = 7.60 Hz, 1H), 7.35 (t, J = 7.80 Hz, 1H), 7.23 (s, 1H), 2.93 (t, J = 4.60 Hz, 4H), 1.63 (quin, J = 5.50 Hz, 4H), 1.37 - 1.46 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.8, 159.1, 149.8, 139.6, 134.7, 133.6, 131.7, 130.7, 129.1, 128.3, 127.7, 127.5, 127.5, 126.2, 126.2, 125.5, 125.2, 112.3, 46.8, 25.1, 23.4. HRMS for C₂₅H₂₄N₃O₃S₂ [M + H⁺] calculated 478.1254, found 478.1250.

Compounds 12r-s were synthesized using the same protocol as for compound 1.

N-(5-methyl-8H-indeno[1,2-d]thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12r).

2,3-dihydro-1H-inden-1-one (13r, 250 mg, 1.71 mmol) was converted to compound 14r which was followed by ‘HATU coupling’ reaction with compound 4 (29 mg, 0.11 mmol) to obtain 12r (12 mg, yield = 25% for the final step) as light yellow solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.24 (br. s., 1H), 8.10 - 8.14 (m, 2H), 7.69 (d, J = 8.31 Hz, 2H), 7.35 (d, J = 7.83 Hz, 1H), 6.97 (d, J = 7.58 Hz, 1H), 6.86 (s, 1H), 3.85 (s, 2H), 2.79 (t, J = 4.52 Hz, 4H), 2.24 (s, 3H), 1.60 (quin, J = 5.69 Hz, 4H), 1.37 - 1.44 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.9, 162.6, 155.3, 142.7, 140.3, 136.3, 136.2, 135.7, 131.6, 128.4, 128.0, 126.2, 124.6, 119.0, 46.6, 32.3, 25.1, 23.2, 21.4. HRMS for C₂₃H₂₄N₃O₃S₂ [M + H]⁺ calculated 454.1254, found 454.1248.

N-(8-methyl-4,5-dihydronaphtho[1,2-d]thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12s).

3,4-dihydronaphthalen-1(2H)-one (13s, 125 mg, 0.78 mmol) was converted to compound 14s which was followed by ‘HATU coupling’ reaction with compound 4 (27 mg, 0.1 mmol) to obtain 12s (25 mg, yield = 49% for the final step) as light yellow solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.54 (br. s., 1H), 7.94 (d, J = 8.56 Hz, 2H), 7.65 (d, J = 8.31 Hz, 2H), 7.26 (s, 1H), 7.07 (d, J = 7.58 Hz, 1H), 6.92 (dd, J = 0.98, 7.58 Hz, 1H), 2.99 - 3.03 (m, 4H), 2.90 (t, J = 5.38 Hz, 4H), 2.21 (s, 3H), 1.63 (td, J = 5.78, 11.19 Hz, 4H), 1.39 - 1.45 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.9, 156.8, 144.2, 139.9, 136.2, 135.8, 131.7, 130.1, 128.0, 128.0, 127.9, 127.7, 125.3, 122.9, 46.8, 28.5, 25.1, 23.3, 21.6, 21.2. HRMS for C₂₄H₂₆N₃O₃S₂ [M + H]⁺ calculated 468.1410, found 468.1404.

N-(4-(5-ethynyl-2-methylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12t).

Copper iodide (3.5 mg, 0.018 mmol), tetrakis(triphenylphosphine)palladium(0) (26 mg, 0.037 mmol) were taken in a microwave vial and then the vial was evacuated under vacuum and flushed with argon. Trimethylsilylacetylene (318 mg, 3.23 mmol) and diethylamine (1.5 mL) was then added to the vial. Separately, compound 8e (100 mg, 0.37 mmol) was dissolved in anhydrous DMF and added to the vial. The reaction was then performed on microwave at 100 °C for 1h. The solvent was then removed and the residue was dissolved in EtOAc, washed with water and brine, dried over magnesium sulfate and concentrated under vacuum to obtain the residue which was purified using silica gel column chromatography to obtain TMS protected intermediate (76 mg). To a solution of this intermediate (67 mg, 0.23 mmol) in anhydrous THF was added a 1M solution of tetra-n-butylammonium fluoride in THF (2 mL) and the reaction was stirred till completion. The reaction was poured into water and compound was extracted with EtOAc. The EtOAc fraction was then concentrated under vacuum, dried over magnesium sulfate to obtain residue which was purified using silica gel column chromatography to obtain compound 15 in quantitative yield. To a solution of compound 4 (20 mg, 0.074 mmol) in anhydrous DMF were added, 15 (16 mg, 0.074 mmol), HATU (34 mg, 0.089 mmol), and triethylamine (22 mg, 0.22 mmol) and the reaction was heated at 45 °C for 12h. The solvent was then removed and the residue was suspended in aqueous sodium bicarbonate solution. The precipitate was filtered to obtain crude product which was purified using silica gel column chromatography to yield compound 12t as off-white solid (23 mg, yield = 67%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.17 (br. s., 1H), 7.98 - 8.02 (m, 2H), 7.78 - 7.83 (m, 2H), 7.60 (d, J = 1.71 Hz, 1H), 7.32 (dd, J = 1.71, 7.83 Hz, 1H), 7.17 (d, J = 7.83 Hz, 1H), 7.03 - 7.05 (m, 1H), 3.07 (s, 1H), 3.01 (t, J = 5.38 Hz, 4H), 2.42 (s, 3H), 1.66 (quin, J = 5.62 Hz, 4H), 1.40 - 1.47 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.6, 157.9, 154.4, 148.8, 140.3, 136.9, 135.4, 133.9, 133.1, 131.7, 131.1, 128.1, 128.0, 119.8, 112.1, 83.0, 46.9, 25.1, 23.4, 21.0. HRMS for C₂₄H₂₄N₃O₃S₂ [M + H]⁺ calculated 466.1254, found 466.1248.

N-(4-(5-ethyl-2-methylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (12u).

To a solution of compound 12t (6 mg, 0.013 mmol) in methanol, was added a catalytic amount of palladium on carbon and the flask was subjected to hydrogenation reaction using Parr shaker apparatus for 4h. The solvent was then filtered to obtain the filtrate which was concentrated under vacuum to obtain compound 12u as off-white solid (3 mg, yield = 50%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.87 (d, J = 8.07 Hz, 2H), 7.64 - 7.75 (m, J = 8.07 Hz, 2H), 7.25 (s, 1H), 7.05 (d, J = 7.83 Hz, 1H), 7.01 (s, 1H), 6.97 (d, J = 7.83 Hz, 1H), 2.97 (t, J = 4.89 Hz, 4H), 2.56 (q, J = 7.58 Hz, 2H), 2.35 (s, 3H), 1.64 (td, J = 5.53, 10.70 Hz, 4H), 1.38 - 1.49 (m, 2H), 1.18 (t, J = 7.58 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.0, 158.4, 149.8, 141.7, 139.7, 135.4, 133.3, 132.7, 131.1, 128.6, 128.0, 127.8, 127.6, 111.4, 46.9, 28.2, 25.1, 23.4, 20.5, 15.4. HRMS for C₂₄H₂₈N₃O₃S₂ [M + H]⁺ calculated 470.1567, found 470.1562.

N-(4-(2,5-dimethylphenyl)-1H-imidazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18a).

To a solution of 1-(2,5-dimethylphenyl)ethan-1-one (6a, 200 mg, 1.35 mmol) in chloroform was added liquid bromine (238 mg, 1.49 mmol). The reaction was stirred for an hour, followed by removal of the solvent to obtain crude intermediate which was dissolved in anhydrous EtOH and 1-acetylguanidine (273 mg, 2.7 mmol) was added. The reaction was then heated in a microwave at 120 °C for 1h, followed by removal of the solvent to obtain the residue which was purified using silica gel column chromatography to obtain compound 16 (75 mg, yield = 24%). Compound 16 was then dissolved in 50% MeOH/3M HCl and heated at 55 °C for 1h. The solvent was then removed to obtain compound 17. To a solution of compound 4 (7.5 mg, 0.039 mmol) in anhydrous DMF were added, 17 (10 mg, 0.037 mmol), HATU (16 mg, 0.041 mmol), and triethylamine (8 mg, 0.08 mmol) and the reaction was heated at 45 °C for 12h. The solvent was then removed and the residue was suspended in aqueous sodium bicarbonate solution. The precipitate was filtered to obtain crude product which was purified using silica gel column chromatography to yield compound 18a as tan solid (4.2 mg, yield = 26%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.37 - 8.43 (m, 2H), 7.93 - 7.98 (m, 2H), 7.19 - 7.26 (m, 3H), 7.03 (s, 1H), 5.35 (br. s., 1H), 3.04 - 3.09 (m, 4H), 2.42 (s, 3H), 2.41 (s, 3H), 1.66 - 1.69 (m, 4H), 1.44 - 1.48 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 153.3, 141.4, 136.6, 134.1, 132.9, 131.5, 130.8, 129.4, 128.2, 125.4, 47.0, 29.7, 25.2, 23.4, 20.9, 20.4. HRMS for C₂₃H₂₇N₄O₃S [M + H]⁺ calculated 439.1798, found 439.1796.

Compounds 18b-d were synthesized using similar protocol as shown below for 18b. Briefly, Suzuki coupling reaction between 19 and 20b-d yielded 21b-d which was followed by ‘HATU coupling’ reaction with compound 4 as described for synthesis of compound 1.

N-(6-(2,5-dimethylphenyl)pyridin-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18b).

2,5-dimethylphenylboronic acid (19, 50 mg, 0.33 mmol), tetrakis(triphenylphosphine)palladium(0) (65 mg, 0.056 mmol) and 2-amino-6-bromopyridine (20b, 48 mg, 0.28 mmol) were taken in a microwave vial. The vial was evacuated under vacuum and anhydrous DMF (1.6 mL) was added. Separately sodium carbonate (89 mg, 0.84 mmol) was dissolved in 0.4 mL of water and added to the vial. The vial was then subjected to microwave heating at 110 °C for 30 min. The solvent was removed under vacuum and the residue was dissolved in EtOAc, washed with water and brine, concentrated under vacuum to obtain the residue which was purified using silica gel column chromatography to obtain compound 21b. To a solution of compound 4 (15 mg, 0.056 mmol) in anhydrous DMF were added, 21b (12 mg, 0.061 mmol), HATU (23 mg, 0.061 mmol), and triethylamine (8.5 mg, 0.084 mmol) and the reaction was heated at 45 °C for 12h. The solvent was then removed and the residue was suspended in aqueous sodium bicarbonate solution. The precipitate was filtered to obtain crude product which was purified using silica gel column chromatography to yield compound 18b as white solid (14 mg, yield = 56%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.74 (br. s., 1H), 8.34 (d, J = 8.31 Hz, 1H), 8.06 (d, J = 8.07 Hz, 2H), 7.83 - 7.91 (m, 3H), 7.21 (d, J = 7.58 Hz, 1H), 7.18 (d, J = 6.85 Hz, 2H), 7.14 (d, J = 7.58 Hz, 1H), 3.02 (t, J = 4.90 Hz, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 1.62 - 1.71 (m, 4H), 1.38 - 1.53 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.3, 158.8, 150.4, 139.7, 139.2, 138.8, 138.0, 135.5, 132.3, 130.8, 129.9, 129.3, 128.0, 127.8, 120.8, 112.1, 46.9, 25.1, 23.4, 20.9, 19.8. HRMS for C₂₅H₂₈N₃O₃S [M + H]⁺ calculated 450.1846, found 450.1844.

N-(5-(2,5-dimethylphenyl)pyridin-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18c).

18c was synthesized using 19 (50 mg, 0.33 mmol) and 20c (48 mg, 0.28 mmol) to obtain 21c (46 mg). Of which 21 mg of 21c was coupled with compound 4 (25 mg, 0.093 mmol) using ‘HATU coupling’ reaction to obtain compound 18c as off-white solid (18 mg, yield = 43%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.73 (br. s., 1H), 8.43 (d, J = 8.56 Hz, 1H), 8.30 (s, 1H), 8.10 (s, 2H), 7.91 (d, J = 8.31 Hz, 2H), 7.78 (dd, J = 2.20, 8.31 Hz, 1H), 7.21 (d, J = 7.83 Hz, 1H), 7.14 (d, J = 7.58 Hz, 1H), 7.06 (s, 1H), 2.96 - 3.09 (m, 4H), 2.38 (s, 3H), 2.26 (s, 3H), 1.64 - 1.71 (m, 4H), 1.41 - 1.49 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.2, 149.6, 147.9, 139.8, 139.2, 137.9, 137.2, 135.7, 134.5, 132.4, 130.6, 130.5, 128.8, 128.1, 127.8, 113.4, 46.9, 25.1, 23.4, 20.9, 19.9. HRMS for C₂₅H₂₈N₃O₃S [M + H]⁺ calculated 450.1846, found 450.1843.

N-(6-(2,5-dimethylphenyl)pyridin-3-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18d).

18d was synthesized using 19 (50 mg, 0.33 mmol) and 20d (48 mg, 0.28 mmol) to obtain 21d. Of which 25 mg of 21d was coupled to compound 4 (20.3 mg, 0.1 mmol) using ‘HATU coupling’ reaction to obtain compound 18d as yellow solid (16 mg, yield = 36%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.87 (d, J = 2.20 Hz, 1H), 8.62 (br. s., 1H), 8.43 (dd, J = 2.32, 8.44 Hz, 1H), 8.03 (d, J = 8.31 Hz, 2H), 7.78 (d, J = 8.31 Hz, 2H), 7.48 (d, J = 8.56 Hz, 1H), 7.23 (s, 1H), 7.15 - 7.19 (m, 1H), 7.10 - 7.14 (m, 1H), 3.01 (t, J = 5.26 Hz, 4H), 2.36 (s, 3H), 2.34 (s, 3H), 1.66 (quin, J = 5.62 Hz, 4H), 1.41 - 1.48 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 165.1, 155.9, 140.7, 139.3, 139.0, 138.3, 135.4, 133.0, 132.5, 130.8, 130.2, 129.2, 128.1, 128.0, 128.0, 124.4, 46.9, 25.1, 23.4, 20.9, 19.9. HRMS for C₂₅H₂₈N₃O₃S [M + H]⁺ calculated 450.1846, found 450.1843.

N-(5-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18e).

2,5-dimethylphenylboronic acid (19, 32 mg, 0.21 mmol), tetrakis(triphenylphosphine)palladium(0) (104 mg, 0.09 mmol) and compound 9 (50 mg, 0.18 mmol) were taken in a microwave vial. The vial was evacuated under vacuum and anhydrous DMF (1.6 mL) was added. Separately sodium carbonate (57 mg, 0.54 mmol) was dissolved in 0.4 mL of water and added to the vial. The vial was then subjected to microwave heating at 110 °C for 60 min. The solvent was removed under vacuum and the residue was dissolved in EtOAc, washed with water and brine, concentrated under vacuum to obtain the residue which was purified using silica gel column chromatography to obtain N-Boc protected intermediate (15 mg), which was stirred in 4N HCl solution in dioxane for 1h. The solvent was then removed to obtain compound 22. To a solution of compound 4 (16 mg, 0.059 mmol) in anhydrous DMF were added, 22 (10 mg, 0.05 mmol), HATU (38 mg, 0.1 mmol), and DIPEA (14 mg, 0.1 mmol) and the reaction was heated at 45 °C for 12h. The solvent was then removed and the residue was suspended in aqueous sodium bicarbonate solution. The precipitate was filtered to obtain crude product which was purified using silica gel column chromatography to yield compound 18e as off-white solid (6 mg, yield = 27%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.23 (d, J = 8.07 Hz, 2H), 7.93 (d, J = 8.31 Hz, 2H), 7.08 - 7.22 (m, 4H), 3.02 (t, J = 5.14 Hz, 4H), 2.38 (s, 3H), 2.36 (s, 3H), 1.63 (quin, J = 5.60 Hz, 4H), 1.37 - 1.45 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.3, 159.2, 140.4, 136.3, 135.9, 134.4, 133.1, 132.6, 131.0, 130.9, 129.7, 129.3, 128.8, 128.0, 46.9, 25.1, 23.4, 20.8, 20.8. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H]⁺ calculated 456.1410, found 456.1406.

N-(5-(2,5-dimethylphenyl)-1,3,4-thiadiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18f).

2,5-dimethylphenylboronic acid (23, 200 mg, 1.33 mmol) and thiosemicarbazide (182 mg, 2 mmol) were dissolved in 2 mL of phosphorous(V) oxychloride and heated in microwave at 105 °C for 1h. The solvent was then removed and the residue was purified using silica gel column chromatography to obtain compound 24. To a solution of compound 4 (20 mg, 0.074 mmol) in anhydrous DMF were added, 24 (15 mg, 0.074 mmol), HATU (34 mg, 0.09 mmol), and triethylamine (22 mg, 0.22 mmol) and the reaction was heated at 45 °C for 12h. The solvent was then removed and the residue was suspended in aqueous sodium bicarbonate solution. The precipitate was filtered to obtain crude product which was purified using silica gel column chromatography to yield compound 18f as tan solid (17 mg, yield = 50%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 13.36 (br. s., 1H), 8.51 - 8.56 (m, 2H), 7.90 - 7.94 (m, 2H), 7.54 (s, 1H), 7.20 - 7.25 (m, 2H), 2.93 - 3.00 (m, 4H), 2.53 (s, 3H), 2.42 (s, 3H), 1.57 - 1.61 (m, 4H), 1.36 - 1.42 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.4, 163.3, 161.4, 140.6, 136.2, 134.8, 133.8, 131.6, 131.2, 130.8, 129.5, 128.8, 128.0, 46.9, 25.1, 23.4, 21.1, 20.9. HRMS for C₂₂H₂₅N₄O₃S₂ [M + H]⁺ calculated 457.1363, found 457.1363.

5-bromo-4-(2,5-dimethylphenyl)thiazol-2-amine (25).

To a solution of compound 8a (1 g, 4.9 mmol) in 50 mL of dichloromethane was slowly added a solution of N-bromosuccinimide (1.13 g, 6.35 mmol) in 20 mL of dichloromethane and the reaction was stirred for 30 minutes. The solvent was then removed and the residue was purified using column chromatography to obtain compound 25 (750 mg, 55%). MS for C₁₁H₁₁BrN₂S [M + H]⁺ calculated 283.0, found 282.9.

N-(5-(diethylamino)-4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18g).

Compound 25 (25 mg, 0.088 mmol), 1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl₂, 6.5 mg, 0.01 mmol) were taken in a microwave vial, sealed, evacuated under vacuum and purged with argon. Diethylamine (646 mg, 8.8mmol) and 0.9 mL of dioxane was then added to the vial and heated in microwave at 100 °C for 1.5h. The solvent was then removed and the residue was extracted in EtOAc, washed with water and brine, concentrated under vacuum to obtain a residue which was purified using column chromatography to obtain compound 26g (7.2 mg, yield = 30%). The ‘HATU coupling’ reaction was then performed using 4 (5 mg, 0.019 mmol), 26g (5 mg, 0.018 mmol), HATU (7.6 mg, 0.02 mmol), and DIPEA (4.7 mg, 0.036 mmol) to obtain compound 18g as yellow solid (5.6 mg, yield = 62%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.25 (d, J = 8.07 Hz, 2H), 7.91 (d, J = 7.83 Hz, 2H), 7.09 - 7.18 (m, 3H), 3.04 (t, J = 5.26 Hz, 4H), 2.97 (q, J = 7.09 Hz, 4H), 2.34 (s, 3H), 2.23 (s, 3H), 1.64 - 1.68 (m, 4H), 1.45 (d, J = 4.89 Hz, 2H), 1.05 (t, J = 7.09 Hz, 6H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.7, 163.9, 154.0, 153.3, 141.9, 140.7, 135.2, 134.8, 134.1, 131.0, 130.4, 130.0, 128.8, 128.1, 49.6, 46.9, 25.1, 23.4, 20.9, 19.5, 12.6. HRMS for C₂₇H₃₅N₄O₃S₂ [M + H]⁺ calculated 527.2145, found 527.2143.

N-(4-(2,5-dimethylphenyl)-5-(piperidin-1-yl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18h).

To a solution of piperidine (24 mg, 0.28 mmol) in anhydrous DMF, was added 60% dispersion of sodium hydride in mineral oil (22 mg, 0.56 mmol). Compound 25 (40 mg, 0.14 mmol) was separately dissolved in anhydrous DMF and added to above reaction. The reaction was stirred for 1h, followed by removal of the solvent. The residue was dissolved in EtOAc, washed with water, brine and concentrated under vacuum to obtain residues which was purified using silica gel column chromatography to obtain compound 26h (55.7 mg, yield = 15%). The ‘HATU coupling’ reaction was then performed using 4 (6 mg, 0.022 mmol), 26h (5.7 mg, 0.02 mmol), HATU (9.1 mg, 0.024 mmol), and triethylamine (3 mg, 0.03 mmol) to obtain compound 18h as yellow solid (4 mg, yield = 37%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.44 (br. s., 1H), 7.94 (d, J = 8.31 Hz, 2H), 7.79 (d, J = 8.31 Hz, 2H), 7.11 (s, 1H), 7.03 (d, J = 7.58 Hz, 1H), 6.95 (d, J = 7.83 Hz, 1H), 3.01 (t, J = 5.38 Hz, 4H), 2.81 - 2.86 (m, 4H), 2.29 (s, 3H), 2.25 (s, 3H), 1.66 (td, J = 5.78, 11.19 Hz, 4H), 1.55 (td, J = 5.53, 10.70 Hz, 4H), 1.41 - 1.50 (m, 4H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.0, 152.0, 145.3, 139.8, 137.4, 135.9, 134.7, 133.9, 133.6, 130.4, 130.3, 128.5, 127.9, 127.9, 54.8, 46.9, 25.9, 25.1, 23.6, 23.4, 20.9, 19.7. HRMS for C₂₈H₃₅N₄O₃S₂ [M + H]⁺ calculated 539.2145, found 539.2145.

N-(4-(2,5-dimethylphenyl)-5-phenylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18i).

Phenylboronic acid (26 mg, 0.22 mmol), tetrakis(triphenylphosphine)palladium(0) (102 mg, 0.088 mmol) and compound 25 (50 mg, 0.18 mmol) were taken in a microwave vial. The vial was evacuated under vacuum and anhydrous DMF (1.6 mL) was added. Separately sodium carbonate (56 mg, 0.53 mmol) was dissolved in 0.4 mL of water and added to the vial. The vial was then subjected to microwave heating at 110 °C for 60 min. The solvent was removed under vacuum and the residue was dissolved in EtOAc, washed with water and brine, concentrated under vacuum to obtain the residue which was purified using silica gel column chromatography to obtain compound 26i (10.4 mg, yield = 21%). The ‘HATU coupling’ reaction was then performed using 4 (7.7 mg, 0.029 mmol), 26i (7.3 mg, 0.029 mmol), HATU (19.8 mg, 0.052 mmol), and DIPEA (7.4 mg, 0.058 mmol) to obtain compound 18i as white solid (6.7 mg, yield = 43%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.60 (br. s., 1H), 7.92 (d, J = 8.31 Hz, 2H), 7.74 (d, J = 8.31 Hz, 2H), 7.24 (s, 5H), 6.99 (s, 1H), 6.84 - 6.91 (m, 2H), 2.99 (t, J = 5.26 Hz, 4H), 2.20 (s, 3H), 1.85 (s, 3H), 1.63 - 1.67 (m, 4H), 1.39 - 1.46 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.5, 156.5, 144.6, 140.0, 135.3, 135.1, 133.7, 133.3, 131.8, 130.5, 130.5, 129.3, 128.7, 128.7, 128.1, 127.9, 127.7, 127.6, 46.9, 25.1, 23.4, 20.8, 19.2. HRMS for C₂₉H₃₀N₃O₃S₂ [M + H]⁺ calculated 532.1723 found 532.1717.

N-(4-(2,5-dimethylphenyl)-5-(furan-2-yl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18j).

Compound 18j was synthesized using the same procedure as 18i starting with 2-furanylboronic acid (24 mg, 0.21 mmol) and compound 25 (50 mg, 0.18 mmol) to obtain compounds 26j (9.7 mg, yield = 17%). The ‘HATU coupling’ reaction was then performed using 4 (6 mg, 0.022 mmol), 26j (5 mg, 0.019 mmol), HATU (14 mg, 0.037 mmol), and DIPEA (5.3 mg, 0.041 mmol) to obtain compound 18j as tan solid (2.7 mg, yield = 27%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.52 (br. s., 1H), 8.03 (d, J = 8.56 Hz, 2H), 7.85 (d, J = 8.31 Hz, 2H), 7.35 - 7.39 (m, 1H), 7.04 - 7.13 (m, 3H), 6.30 (dd, J = 1.96, 3.42 Hz, 1H), 5.82 (d, J = 3.42 Hz, 1H), 3.02 (t, J = 5.26 Hz, 4H), 2.30 (s, 3H), 2.05 (s, 3H), 1.66 (td, J = 5.75, 11.00 Hz, 4H), 1.44 (quin, J = 5.69 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.0, 155.6, 146.7, 141.9, 140.4, 135.5, 135.2, 133.8, 133.7, 130.4, 130.1, 129.7, 128.1, 128.1, 119.6, 116.0, 111.8, 107.0, 46.9, 25.1, 23.4, 20.9, 18.9. HRMS for C₂₇H₂₈N₃O₄S₂ [M + H]⁺ calculated 522.1516, found 522.1513.

N-(4-(2,5-dimethylphenyl)-5-(ethylthio)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18k).

To a solution of compound 25 (20 mg, 0.071 mmol) in anhydrous DMF, was added sodium ethanthiolate (9 mg, 0.11 mmol) and stirred for 1h. The solvent was then removed and the residue was dissolved in EtOAc, washed with water and brine, concentrated under vacuum to obtain the residue which was purified using silica gel column chromatography to obtain compound 26k (10.3 mg, yield = 55%). The ‘HATU coupling’ reaction was then performed using 4 (12.6 mg, 0.047 mmol), 26k (10.3 mg, 0.039 mmol), HATU (30 mg, 0.079 mmol), and DIPEA (11.1 mg, 0.087 mmol) to obtain compound 18k as yellow solid (9.9 mg, yield = 49%). 1H NMR (500 MHz, CHLOROFORM-d) δ 8.29 - 8.33 (m, 2H), 7.90 - 7.95 (m, 2H), 7.18 (d, J = 1.22 Hz, 2H), 7.04 (s, 1H), 3.03 - 3.07 (m, 4H), 2.71 - 2.77 (m, 2H), 2.35 (s, 3H), 2.20 (s, 3H), 1.66 (quin, J = 5.62 Hz, 4H), 1.42 - 1.48 (m, 2H), 1.23 (t, J = 7.34 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.5, 160.7, 147.0, 141.1, 135.3, 134.0, 133.9, 131.1, 131.0, 130.6, 129.1, 128.7, 128.1, 121.0, 46.9, 31.8, 25.1, 23.4, 20.9, 19.4, 14.7. HRMS for C₂₅H₃₀N₃O₃S₃ [M + H]⁺ calculated 516.1444, found 516.1439.

N-(4-(2,5-dimethylphenyl)-5-ethoxythiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18l).

To a cooled solution of sodium ethoxide (12 mg, 0.18 mmol) in anhydrous ethanol was added a solution of compound 25 (25 mg, 0.088 mmol) in anhydrous ethanol. The reaction was stirred for 1h followed by removal of solvent under vacuum. The residue was dissolved in EtOAc, washed with water and brine, dried over sodium sulfate, concentrated under vacuum to obtain residue which was purified using C18 reverse phase column chromatography to obtain compound 26l (4.9 mg, yield = 23%). The ‘HATU coupling’ reaction was then performed using 4 (5.3 mg, 0.02 mmol), 26l (4.9 mg, 0.02 mmol), HATU (8.25 mg, 0.022 mmol), and DIPEA (5.1 mg, 0.04 mmol) to obtain compound 18l as yellow solid (3.6 mg, yield = 36%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.01 (br. s., 1H), 7.90 (d, J = 8.31 Hz, 2H), 7.75 (d, J = 8.07 Hz, 2H), 7.06 (s, 1H), 7.01 (d, J = 7.83 Hz, 1H), 6.93 (d, J = 7.83 Hz, 1H), 4.09 (q, J = 7.09 Hz, 2H), 2.94 - 3.03 (m, 4H), 2.26 (s, 3H), 2.21 (s, 3H), 1.64 (td, J = 5.72, 10.82 Hz, 4H), 1.40 - 1.46 (m, 2H), 1.32 - 1.36 (m, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.2, 150.4, 146.5, 139.9, 135.4, 134.9, 133.7, 131.8, 130.9, 130.4, 130.4, 128.9, 128.0, 127.8, 72.3, 46.9, 25.1, 23.4, 20.9, 19.6, 15.0. HRMS for C₂₅H₃₀N₃O₄S₂ [M + H]⁺ calculated 500.1672, found 500.1669.

N-(4-(2,5-dimethylphenyl)-5-methylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18m).

Compound 18m was synthesized using the same procedure as for compound 1. 1-(2,5-dimethylphenyl)propan-1-one (27, 100 mg, 0.62 mmol) was converted to compound 28 which was followed by ‘HATU coupling’ reaction with compound 4 (20 mg, 0.075 mmol) to obtain 18m (26 mg, yield =74% for the final step) as off-white solid. ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.87 - 7.99 (m, J = 8.31 Hz, 2H), 7.73 - 7.80 (m, J = 8.07 Hz, 2H), 7.00 - 7.06 (m, 1H), 6.98 (s, 1H), 6.90 (s, 1H), 2.99 (t, J = 4.89 Hz, 4H), 2.27 (s, 3H), 2.31 (s, 3H), 2.10 (s, 3H), 1.57 - 1.69 (m, 4H), 1.33 - 1.50 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.6, 155.5, 145.4, 139.7, 135.5, 135.0, 133.7, 132.9, 130.5, 130.3, 129.1, 128.1, 127.7, 123.6, 46.9, 25.1, 23.4, 20.9, 19.2, 11.7. HRMS for C₂₄H₂₈N₃O₃S₂ [M + H⁺] calculated 470.1567 found 470.1562.

N-(5-bromo-4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18n).

The ‘HATU coupling’ reaction was then performed using 4 (250 mg, 0.93 mmol), 25 (263 mg, 0.93 mmol), HATU (423 mg, 1.12 mmol), and triethylamine (141 mg, 1.4 mmol) to obtain compound 18n as tan solid (305 mg, yield = 62%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.21 (br. s., 1H), 7.92 (d, J = 8.31 Hz, 2H), 7.79 (d, J = 8.56 Hz, 2H), 6.98 - 7.13 (m, 3H), 2.97 - 3.12 (m, 4H), 2.28 (s, 3H), 2.15 (s, 3H), 1.61 - 1.71 (m, 4H), 1.43 (d, J = 5.14 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.5, 157.5, 148.4, 140.4, 135.2, 134.5, 133.8, 131.7, 130.4, 130.4, 129.9, 128.1, 127.9, 101.6, 46.9, 25.1, 23.4, 20.9, 19.3. HRMS for C₂₃H₂₅BrN₃O₃S₂ [M + H]⁺ calculated 534.0515, found 534.0510.

N-(4-(2,5-dimethylphenyl)-5-ethynylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18o).

The 5-bromo displacement by alkyne was performed using Sonogashira coupling reaction. Copper iodide (1.37 mg, 0.007 mmol), tetrakis(triphenylphosphine)palladium(0) (10.1 mg, 0.009 mmol) were taken in a microwave vial and then the vial was evacuated under vacuum and flushed with argon. Trimethylsilylacetylene (205 mg, 2.09 mmol) and diethylamine (1.5 mL) was then added to the vial. Separately, compound 18n (77 mg, 0.14 mmol) was dissolved in anhydrous DMF and added to the vial. The reaction was then performed on microwave at 100 °C for 2h. The solvent was then removed and the residue was dissolved in EtOAc, washed with water and brine, dried over magnesium sulfate and concentrated under vacuum to obtain the residue which was purified using silica gel column chromatography to obtain TMS protected intermediate (74 mg, yield = 95%). The trimethylsilyl (TMS) functional group removal was performed by stirring the solution of this intermediate (74 mg, 0.13 mmol) in anhydrous THF and 1M solution of tetra-n-butylammonium fluoride in THF (2 mL) till completion. The reaction was poured into water and compound was extracted with EtOAc. The EtOAc fraction was then concentrated under vacuum, dried over magnesium sulfate to obtain residue which was purified using silica gel column chromatography to obtain compound 18o as orange solid (59 mg, yield = 95%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.27 (br. s., 1H), 7.85 - 7.91 (m, 2H), 7.73 - 7.78 (m, 2H), 7.12 (s, 1H), 7.00 - 7.03 (m, 1H), 6.94 - 6.99 (m, 1H), 3.35 - 3.38 (m, 1H), 2.95 - 3.01 (m, 4H), 2.27 (s, 3H), 2.23 (s, 3H), 1.61 - 1.65 (m, 4H), 1.39 - 1.46 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.6, 156.7, 155.0, 140.4, 135.0, 134.7, 133.6, 132.3, 130.6, 130.3, 129.8, 128.1, 127.9, 109.0, 85.4, 74.3, 46.9, 25.1, 23.4, 20.8, 19.6. HRMS for C₂₅H₂₆N₃O₃S₂ [M + H]⁺ calculated 480.1410, found 480.1405.

Compounds 18p and 18q were synthesized using the same Sonogashira coupling procedure as for compound 18o.

N-(5-(but-1-yn-1-yl)-4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18p).

Sonogashira coupling reaction was performed using copper iodide (1.72 mg, 0.009 mmol), tetrakis(triphenylphosphine)palladium(0) (10.8 mg, 0.009 mmol), 1-butyne (100 mg, 1.85 mmol) obtained by condensing the gas in cold sealed flask, diisopropylamine (1.5 mL) and compound 18n (50 mg, 0.09 mmol) to obtain compound 18p as white solid (12.5 mg, yield = 27%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.06 (br. s., 1H), 7.90 (d, J = 8.31 Hz, 2H), 7.77 (d, J = 8.31 Hz, 2H), 7.17 (s, 1H), 7.00 - 7.05 (m, 1H), 6.95 - 6.99 (m, 1H), 3.00 (t, J = 5.26 Hz, 4H), 2.38 (q, J = 7.34 Hz, 2H), 2.28 (s, 3H), 2.26 (s, 3H), 1.65 (quin, J = 5.69 Hz, 4H), 1.40 - 1.46 (m, 2H), 1.13 - 1.19 (m, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.4, 155.3, 152.3, 140.2, 135.0, 134.8, 133.6, 132.8, 130.5, 130.5, 129.4, 128.0, 127.9, 111.1, 100.1, 70.5, 46.9, 25.1, 23.4, 20.8, 19.7, 13.6, 13.5. HRMS for C₂₇H₃₀N₃O₃S₂ [M + H]⁺ calculated 508.1723, found 508.1719.

N-(4-(2,5-dimethylphenyl)-5-(pent-1-yn-1-yl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18q).

Sonogashira coupling reaction was performed using copper iodide (1.03 mg, 0.005 mmol), tetrakis(triphenylphosphine)palladium(0) (6.5 mg, 0.005 mmol), 1-pentyne (11.5 mg, 0.17 mmol), diisopropylamine (0.45 mL) and compound 18n (30 mg, 0.05 mmol) to obtain compound 18q as light yellow solid (20.5 mg, yield = 78%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.05 (br. s., 1H), 7.89 (d, J = 8.31 Hz, 2H), 7.76 (d, J = 8.31 Hz, 2H), 7.16 (s, 1H), 7.00 - 7.03 (m, 1H), 6.94 - 6.98 (m, 1H), 2.99 (t, J = 5.26 Hz, 4H), 2.34 (t, J = 6.97 Hz, 2H), 2.27 (s, 3H), 2.24 (s, 3H), 1.62 - 1.67 (m, 4H), 1.54 (sxt, J = 7.24 Hz, 2H), 1.39 - 1.45 (m, 2H), 0.94 (t, J = 7.34 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.4, 155.3, 152.3, 140.1, 135.0, 134.8, 133.6, 132.8, 130.5, 130.5, 129.4, 128.0, 127.9, 111.2, 98.7, 71.2, 46.9, 25.1, 23.4, 21.8, 21.8, 20.8, 19.7, 13.4. HRMS for C₂₈H₃₂N₃O₃S₂ [M + H]⁺ calculated 522.1880, found 522.1878.

N-(4-(2,5-dimethylphenyl)-5-ethylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18r).

To a solution of compound 18o (59 mg, 0.12 mmol) in methanol was added a catalytic amount of palladium on carbon. The reaction was subjected to hydrogenation on a Parr hydrogenation apparatus using hydrogen gas at 50 psi pressure for 6h. The solvent was then removed, and the residue was purified using silica gel column chromatography to obtain compound 18r as tan solid (24 mg, yield = 42%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.97 (d, J = 8.31 Hz, 2H), 7.79 (d, J = 8.31 Hz, 2H), 7.05 (d, J = 7.83 Hz, 1H), 6.99 (d, J = 7.58 Hz, 1H), 6.90 (s, 1H), 3.00 (t, J = 5.01 Hz, 4H), 2.68 (q, J = 7.50 Hz, 2H), 2.28 (s, 3H), 2.09 (s, 3H), 1.65 (quin, J = 5.50 Hz, 4H), 1.37 - 1.48 (m, 2H), 1.26 (t, J = 7.46 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.5, 155.3, 151.3, 139.9, 135.7, 135.1, 133.8, 133.4, 131.4, 130.5, 130.2, 129.1, 128.1, 127.9, 46.9, 25.1, 23.4, 20.9, 20.1, 19.2, 16.4. HRMS for C₂₅H₃₀N₃O₃S₂ [M + H]⁺ calculated 484.1723, found 484.1719.

Compound 18s and 18t were synthesized using the same hydrogenation procedure as for 18r.

N-(5-butyl-4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18s).

Compound 18p (6 mg, 0.012 mmol) was subjected to hydrogenation on Parr hydrogenation apparatus to obtain compound 18s as white solid (4 mg, yield = 65%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.15 (br. s., 1H), 7.93 (d, J = 8.56 Hz, 2H), 7.77 (d, J = 8.31 Hz, 2H), 7.02 - 7.05 (m, 1H), 6.95 - 6.99 (m, 1H), 6.87 (s, 1H), 2.96 - 3.02 (m, 4H), 2.64 (t, J = 7.58 Hz, 2H), 2.27 (s, 3H), 2.06 (s, 3H), 1.57 - 1.67 (m, 6H), 1.39 - 1.45 (m, 2H), 1.27 - 1.35 (m, 2H), 0.85 (t, J = 7.46 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.5, 155.5, 144.9, 139.8, 135.7, 135.0, 133.9, 133.4, 130.5, 130.2, 129.9, 129.1, 128.1, 127.8, 46.9, 33.8, 26.1, 25.1, 23.4, 22.1, 20.9, 19.2, 13.7. HRMS for C₂₇H₃₄N₃O₃S₂ [M + H]⁺ calculated 512.2036, found 512.2032.

N-(4-(2,5-dimethylphenyl)-5-pentylthiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (18t).

Compound 18q (10 mg, 0.019 mmol) was subjected to hydrogenation Parr hydrogenation apparatus to obtain compound 18t as white solid (6 mg, yield = 60%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.93 (br. s., 1H), 7.95 (d, J = 8.31 Hz, 2H), 7.79 (d, J = 8.56 Hz, 2H), 7.05 (d, J = 7.83 Hz, 1H), 6.96 - 7.02 (m, 1H), 6.89 (s, 1H), 2.96 - 3.04 (m, 4H), 2.63 (t, J = 7.70 Hz, 2H), 2.28 (s, 3H), 2.07 (s, 3H), 1.58 - 1.68 (m, 8H), 1.40 - 1.46 (m, 2H), 1.22 - 1.28 (m, 5H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.5, 153.7, 150.8, 139.9, 135.7, 135.1, 133.9, 133.5, 130.6, 130.2, 130.0, 129.1, 128.1, 127.9, 46.9, 31.4, 31.2, 26.4, 25.1, 23.4, 22.3, 20.9, 19.2, 13.9. HRMS for C₂₈H₃₆N₃O₃S₂ [M + H]⁺ calculated 526.2193, found 526.2192.

Compound 34a was synthesized using two advanced intermediates 31 and 33 which were synthesized as shown below.

(4-(piperidin-1-ylsulfonyl)phenyl)methanamine (31).

To a solution of 4-cyanobenzenesulfonyl chloride (29, 200 mg, 0.995 mmol) in anhydrous DMF were added potassium carbonate (275 mg, 1.99 mmol) and piperidine (3, 93 mg, 1.095 mmol). The reaction mixture was stirred for an hour followed by removal of the solvent to obtain the residue which was purified using silica gel column chromatograph to obtain compound 30 (102 mg, yield = 41%). To a solution of compound 30 (100 mg, 0.4 mmol) in methanol was added di-tert-butyl dicarbonate (131 mg, 0.6 mmol) and a catalytic amount of palladium on carbon. The reaction was subjected to hydrogenation on a Parr hydrogenation apparatus using hydrogen gas at 50 psi pressure for 4h. The solvent was then removed, and the residue was purified using silica gel column chromatography to obtain compound the N-Boc protected intermediate which was dissolved in 4N HCl/dioxane solution and stirred for 1h to obtain 31 as white solid (40 mg, yield = 35%).

2-bromo-4-(2,5-dimethylphenyl)thiazole (33).

To a solution of compound 6a (16 mg, 0.11 mmol) in chloroform was added liquid bromine (26 mg, 0.165 mmol). The reaction was stirred for an hour, followed by removal of the solvent to obtain crude intermediate 7a which was dissolved in ethanol and potassium thiocyanate (16 mg, 0.16 mmol) was added. The reaction was heated in microwave at 80 °C for 30 min. The solvent was then removed under vacuum and water was added to precipitate the product which was isolated by filtration to obtain compound 32. To the solution of compound 32 (28 mg, 0.14 mmol) in acetic acid (0.5 mL) was 0.5 mL of HBr solution in acetic acid. The reaction was heated at 130 °C for 1h, followed by removal of the solvent. The product was then precipitated in water and collected by filtration to obtain compound 33 (12.5 mg, yield = 33%).

4-(2,5-dimethylphenyl)-N-(4-(piperidin-1-ylsulfonyl)benzyl)thiazol-2-amine (34a).

Compound 34a was then obtained by ‘Buckwald-Hartwig coupling’ reaction using compounds 31 and 33. Compound 31 (15 mg, 0.05 mmol), compound 33 (12.5 mg, 0.047 mmol), bis(dibenzylideneacetone)palladium(0) (Pd(dba)2, 8 mg, 0.014 mmol), (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP, 15 mg, 0.023 mmol) and sodium tert-butoxide (14 mg, 0.14 mmol) were combined in a microwave vial, sealed and air was evacuated under vacuum. Dioxane (0.5 mL) was then added and the reaction was heated in microwave at 100 °C for 1h. The solvent was then removed and the residue was dissolved in EtOAc, washed with water and brine, dried over sodium sulfate, concentrated under vacuum to obtain residues which was purified using silica gel column chromatography to obtain compound 34a a tan solid (4 mg, yield = 18%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.74 (d, J = 8.31 Hz, 2H), 7.56 (d, J = 8.07 Hz, 2H), 7.36 (s, 1H), 7.09 - 7.13 (m, 1H), 7.02 - 7.06 (m, 1H), 6.47 (s, 1H), 5.72 (br. s, 1H), 4.64 (d, J = 4.89 Hz, 2H), 2.96 - 3.01 (m, 4H), 2.36 (s, 3H), 2.33 (s, 3H), 1.62 - 1.68 (m, 4H), 1.42 (t, J = 5.62 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.5, 159.0, 148.0, 143.9, 135.5, 132.9, 132.3, 130.8, 130.2, 129.4, 128.3, 128.1, 127.8, 110.7, 44.8, 33.0, 29.7, 25.6, 20.9, 20.3. HRMS for C₂₃H₂₈N₃O2S₂ [M + H]⁺ calculated 442.1617, found 442.1616.

Compound 34b was synthesized using two advanced intermediates 37 and 39, which were synthesized as shown below.

4-(piperidin-1-ylsulfonyl)aniline (37).

To a solution of 4-nitrobenzenesulfonyl chloride (35, 200 mg, 0.91 mmol) in anhydrous DMF were added potassium carbonate (250 mg, 1.81 mmol) and piperidine (3, 85 mg, 0.995 mmol). The reaction mixture was stirred for an hour followed by removal of the solvent to obtain the residue which was precipitated in water to obtain compound 36 (84 mg, yield = 34%) by filtration. To a solution of compound 36 (80 mg, 0.3 mmol) in 50% EtOAc/methanol was added a catalytic amount of palladium on carbon. The reaction was subjected to hydrogenation on a Parr hydrogenation apparatus using hydrogen gas at 50 psi pressure for 4h. The solvent was then removed, and the residue was purified using silica gel column chromatography to obtain compound the 37 as tan solid (42 mg, yield = 58%).

4-(2,5-dimethylphenyl)thiazole-2-carboxylic acid (39).

To a solution of compound 6a (100 mg, 0.68 mmol) in chloroform was added liquid bromine (118 mg, 0.74 mmol). The reaction was stirred for an hour, followed by removal of the solvent to obtain crude intermediate 7a which was dissolved ethanol and ethyl thiooxamate (98 mg, 0.79 mmol) was added. The reaction was heated in microwave at 120 °C for 30 min. The solvent was then removed under vacuum and water was added to precipitate the product which was isolated by filtration to obtain compound 38 (101 mg, yield = 59%). To the solution of compound 38 (96 mg, 0.37 mmol) in THF was added an aqueous solution of LiOH (46 mg, 1.1 mmol). The reaction was stirred for 12h, followed by removal of solvent under vacuum. Water was then added and the solution was acidified to precipitate the product which was collected by filtration to obtain compound 39 as yellow solid (71 mg, yield = 82%).

4-(2,5-dimethylphenyl)-N-(4-(piperidin-1-ylsulfonyl)phenyl)thiazole-2-carboxamide (34b).

Compound 34a was then obtained by the ‘HATU coupling’ reaction using compounds 37 (17.3 mg, 0.07 mmol), 39 (16 mg, 0.069 mmol), HATU (31 mg, 0.082 mmol) and triethylamine (14 mg, 0.14 mmol) as off-white solid (6.4 mg, yield = 20%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 9.34 (s, 1H), 7.88 - 7.92 (m, 2H), 7.77 - 7.81 (m, 2H), 7.61 (s, 1H), 7.40 (s, 1H), 7.22 - 7.25 (m, 1H), 7.17 - 7.20 (m, 1H), 2.98 - 3.02 (m, 4H), 2.42 (s, 3H), 2.41 (s, 3H), 1.66 (quin, J = 5.69 Hz, 4H), 1.40 - 1.46 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 161.7, 157.6, 157.0, 140.9, 135.8, 133.1, 133.1, 131.7, 131.0, 130.4, 129.8, 129.1, 122.8, 119.5, 47.0, 25.1, 23.5, 20.9, 20.5. HRMS for C₂₃H₂₆N₃O₃S₂ [M + H⁺] calculated 456.1410, found 456.1407.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-N-methyl-4-(piperidin-1-ylsulfonyl)benzamide (34c).

To a solution of compound 1 (17 mg, 0.037 mmol) in anhydrous DMF were added 60% suspension of sodium hydride in mineral oil (1.6 mg, 0.04 mmol) and iodomethane (6.3 mg, 0.044 mmol). The reaction was stirred for 1h, followed by removal of solvent under vacuum to obtain residue which was purified using column chromatography to obtain compound 34c as off-white solid (7.5 mg, yield = 43%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.47 - 8.53 (m, 2H), 7.83 (d, J = 8.56 Hz, 2H), 7.24 (s, 2H), 7.07 (s, 1H), 6.57 (s, 1H), 3.58 (s, 3H), 3.01 (t, J = 5.38 Hz, 4H), 2.38 (s, 3H), 2.14 (s, 3H), 1.64 (quin, J = 5.62 Hz, 4H), 1.41 (t, J = 5.62 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 172.5, 168.6, 140.9, 139.0, 138.2, 136.0, 134.6, 131.1, 131.0, 130.5, 129.6, 129.6, 127.4, 106.9, 46.9, 34.1, 25.1, 23.5, 20.8, 19.1. HRMS for C₂₄H₂₈N₃O₃S₂ [M + H]⁺ calculated 470.1567, found 470.1564.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)-N-(prop-2-yn-1-yl)benzamide (34d).

To a solution of compound 1 (10 mg, 0.022 mmol) in anhydrous DMF were added 60% suspension of sodium hydride in mineral oil (0.8 mg, 0.033 mmol) 80% propargyl bromide solution in toluene (3.93 mg, 0.026 mmol). The reaction was stirred for 1h, followed by removal of solvent under vacuum to obtain residue which was purified using column chromatography to obtain compound 34d as off-white solid (5.1 mg, yield = 47%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.49 - 8.55 (m, 2H), 7.84 (d, J = 8.07 Hz, 2H), 7.23 - 7.26 (m, 2H), 7.15 (s, 1H), 6.55 (s, 1H), 4.85 (br. s., 1H), 4.70 (br. s., 1H), 3.01 (t, J = 5.26 Hz, 4H), 2.38 (s, 3H), 2.26 (t, J = 2.32 Hz, 1H), 2.19 (s, 3H), 1.63 - 1.67 (m, 4H), 1.38 - 1.45 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 172.7, 168.3, 140.6, 138.4, 137.6, 135.9, 135.1, 131.4, 131.2, 130.6, 129.8, 128.7, 127.4, 107.2, 77.0, 72.6, 47.0, 36.2, 25.1, 23.5, 20.8, 19.4. HRMS for C₁₉H₂₅ClNO₆SNa [M + H]⁺ calculated 494.1567, found 494.1563.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-5-(piperidin-1-ylsulfonyl)picolinamide (42a).

To a solution of methyl 5-(chlorosulfonyl)picolinate (40a, 80 mg, 0.34 mmol) in anhydrous DMF were added, potassium carbonate (70 mg, 0.51 mmol) and piperidine (3, 30 mg, 0.36 mmol). The reaction was stirred for 1h, followed by removal of the solvent. Water was added to precipitate solid which was collected by filtration to obtain methyl ester intermediate (32 mg, yield = 33%) which was dissolved in 1:1 THF/MeOH and aqueous solution of LiOH (14 mg, 0.58 mmol) was added. After stirring for 30 minutes, solvent was removed, water was added and acidified using 3N aqueous HCl solution to precipitate the solid which was collected by filtration to obtain compound 41a (27 mg, yield = 89%). The ‘HATU coupling’ reaction was then performed using 41a (27 mg, 0.1 mmol), 8a (21.5 mg, 0.105 mmol), HATU (42 mg, 0.11 mmol) and triethylamine (22 mg, 0.22 mmol) to obtain compound 42a as yellow solid (39 mg, yield = 85%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.14 (s, 1H), 9.00 (dd, J = 0.73, 2.20 Hz, 1H), 8.45 - 8.50 (m, 1H), 8.26 - 8.32 (m, 1H), 7.44 (s, 1H), 7.18 (d, J = 7.83 Hz, 1H), 7.09 - 7.12 (m, 1H), 7.03 (s, 1H), 3.10 (t, J = 5.38 Hz, 4H), 2.44 (s, 3H), 2.37 (s, 3H), 1.69 (quin, J = 5.69 Hz, 4H), 1.45 - 1.51 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 160.5, 155.7, 150.9, 150.5, 147.2, 137.2, 136.6, 135.4, 134.0, 132.8, 130.8, 130.2, 128.9, 123.0, 111.4, 46.9, 25.1, 23.3, 20.9, 20.7. HRMS for C₂₂H₂₅N₄O₃S₂ [M + H]⁺ calculated 457.1363, found 457.1360.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-6-(piperidin-1-ylsulfonyl)nicotinamide (42b).

Compounds 42b was synthesized using the same procedure as for 42a starting with ethyl 6-(chlorosulfonyl)nicotinate (40b, 80 mg, 0.32 mmol) to obtain intermediate compound 41b (25 mg, yield = 55%). The ‘HATU coupling’ reaction was then performed using 41b (7 mg, 0.026 mmol), 8a (6 mg, 0.029 mmol), HATU (11 mg, 0.026 mmol) and triethylamine (6 mg, 0.057 mmol) to obtain compound 42b as off-white solid (4 mg, yield = 34%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.65 (br. s., 1H), 9.04 (s, 1H), 8.14 (d, J = 8.31 Hz, 1H), 7.79 (d, J = 8.31 Hz, 1H), 7.12 (s, 1H), 6.95 - 7.00 (m, 2H), 6.91 (d, J = 7.83 Hz, 1H), 3.26 (t, J = 5.01 Hz, 4H), 2.28 (s, 3H), 2.24 (s, 3H), 1.60 - 1.64 (m, 4H), 1.50 (d, J = 4.40 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 162.6, 159.3, 155.6, 149.4, 149.0, 136.8, 135.5, 133.0, 132.7, 131.0, 130.0, 129.8, 129.3, 122.3, 111.6, 47.4, 25.4, 23.6, 20.8, 20.1. HRMS for C₂₂H₂₅N₄O₃S₂ [M + H]⁺ calculated 457.1363, found 457.1359.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-5-(piperidin-1-ylsulfonyl)thiophene-2-carboxamide (42c).

To a solution of methyl 5-(chlorosulfonyl)thiophene-2-carboxylate (40c, 25 mg, 0.1 mmol) in anhydrous dichloromethane were added, triethylamine (21 mg, 0.21 mmol) and piperidine (3, 17.5 mg, 0.21 mmol). The reaction was stirred for 1h, followed by removal of the solvent. Water was added to precipitate solid which was collected by filtration to obtain methyl ester intermediate which was dissolved in 1:1 THF/MeOH and aqueous solution of LiOH (7.5 mg, 0.31 mmol) was added. After stirring for 30 minutes, solvent was removed, water was added and acidified using 3N aqueous HCl solution and the product was extracted using EtOAc. The EtOAc fraction was then separated, dried using sodium sulfate, concentrated under vacuum to obtain compound 41c (7 mg, yield = 25%). The ‘HATU coupling’ reaction was then performed using 41c (5.6 mg, 0.021 mmol), 8a (4.3 mg, 0.021 mmol), HATU (10 mg, 0.025 mmol) and triethylamine (3 mg, 0.032 mmol) to obtain compound 42c as white solid (6 mg, yield = 62%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.77 (br. s., 1H), 7.28 - 7.31 (m, 1H), 7.22 - 7.24 (m, 1H), 7.20 (s, 1H), 7.02 - 7.05 (m, 1H), 6.99 (s, 1H), 6.96 - 6.98 (m, 1H), 3.03 (t, J = 5.50 Hz, 4H), 2.31 (s, 3H), 2.29 (s, 3H), 1.66 - 1.71 (m, 4H), 1.44 - 1.49 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 159.1, 152.3, 149.6, 142.4, 141.9, 135.5, 133.3, 132.6, 131.4, 130.9, 130.0, 129.3, 128.8, 111.4, 46.9, 25.0, 23.3, 20.8, 20.2. HRMS for C₂₁H₂₄N₃O₃S₃ [M + H⁺] calculated 462.0974, found 462.0969.

3-chloro-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (42d).

To a solution of 3-chloro-4-(chlorosulfonyl)benzoic acid (40d, 50 mg, 0.2 mmol) in anhydrous DMF were added, potassium carbonate (30 mg, 0.22 mmol) and piperidine (3, 16.7 mg, 0.2 mmol). The reaction was heated at 45 °C for 30 minutes followed by removal of solvent under vacuum to obtain a residue which was purified using C18 reverse phase column chromatography to obtain compound 41d (7.8 mg). The ‘HATU coupling’ reaction was then performed using 41d (7.8 mg, 0.026 mmol), 8a (5.1 mg, 0.026 mmol), HATU (11.6 mg, 0.031 mmol) and triethylamine (5.2 mg, 0.052 mmol) to obtain compound 42d as tan solid (7.9 mg, yield = 62%). ¹H NMR (500 MHz, CHLOROFORM-d) d 7.96 (d, J = 8.07 Hz, 1H), 7.92 (s, 1H), 7.70 (d, J = 7.83 Hz, 1H), 7.18 (s, 1H), 7.02 - 7.05 (m, 1H), 7.00 (s, 1H), 6.94 - 6.99 (m, 1H), 3.26 (t, J = 5.40 Hz, 4H), 2.32 (s, 3H), 2.27 (s, 3H), 1.62 (br. s., 4H), 1.55 (d, J = 4.40 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 162.9, 158.6, 151.1, 149.0, 140.1, 136.1, 135.6, 132.8, 132.6, 131.9, 131.5, 131.1, 129.9, 129.2, 125.3, 111.3, 46.6, 25.5, 23.7, 20.8, 20.3. HRMS for C₂₃H₂₅ClN₃O₃S₂ [M + H]⁺ calculated 490.1020, found 490.1015.

3-bromo-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (42e).

Compound 42e was synthesized using the same procedure as for 42d starting with 3-bromo-4-(chlorosulfonyl)benzoic acid (40a, 48 mg, 0.16 mmol) to obtain compound 41e (8.3 mg, yield = 15%). The ‘HATU coupling’ reaction was then performed using 41e (8.3 mg, 0.024 mmol), 8a (5.4 mg, 0.026 mmol), HATU (11 mg, 0.029 mmol) and triethylamine (4.9 mg, 0.048 mmol) to obtain compound 42e as light yellow solid (5 mg, yield = 39%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.11 (s, 1H), 7.96 (d, J = 8.31 Hz, 1H), 7.72 (d, J = 8.31 Hz, 1H), 7.17 (s, 1H), 6.95 - 7.06 (m, 3H), 3.23 - 3.29 (m, 5H), 2.31 (s, 3H), 2.26 (s, 3H), 1.59 - 1.65 (m, 4H), 1.53 - 1.59 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 162.8, 158.5, 150.0, 141.6, 136.0, 135.5, 135.1, 132.7, 132.6, 132.0, 131.1, 129.8, 129.1, 125.8, 120.8, 111.4, 46.6, 25.5, 23.7, 20.8, 20.4. HRMS for C₂₃H₂₅BrN₃O₃S₂ [M + H]⁺ calculated 534.0515, found 534.0511.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-3-nitro-4-(piperidin-1-ylsulfonyl)benzamide (42f).

Compound 42f was synthesized using the same procedure as for 42c starting with methyl 4-(chlorosulfonyl)-3-nitrobenzoate (40f, 50 mg, 0.18 mmol) to obtain compound 41f (45 mg, yield = 76%). The ‘HATU coupling’ reaction was then performed using 41f (17 mg, 0.054 mmol), 8a (12.2 mg, 0.06 mmol), HATU (25 mg, 0.065 mmol) and triethylamine (10.9 mg, 0.11 mmol) to obtain compound 42f as orange solid (24 mg, yield = 89%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.03 (d, J = 8.31 Hz, 1H), 8.00 - 8.11 (m, 1H), 7.89 (d, J = 8.31 Hz, 1H), 7.15 (s, 1H), 6.95 - 7.07 (m, 3H), 3.20 - 3.38 (m, 4H), 2.32 (s, 3H), 2.26 (s, 3H), 1.65 (br. s., 4H), 1.56 (d, J = 4.40 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 162.2, 147.9, 136.2, 135.7, 135.2, 132.7, 131.2, 129.9, 129.8, 123.8, 111.4, 46.9, 25.4, 23.5, 20.8, 20.2. HRMS for C₂₃H₂₅N₄O₅S₂ [M + H]⁺ calculated 501.1261, found 501.1255.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-3-methoxy-4-(piperidin-1-ylsulfonyl)benzamide (42g).

Compound 42g was synthesized using the same procedure as for 42c starting with methyl 4-(chlorosulfonyl)-3-methoxybenzoate (40g, 50 mg, 0.19 mmol) to obtain compound 41g (47mg, yield = 79%). The ‘HATU coupling’ reaction was then performed using 41g (25 mg, 0.084 mmol), 8a (18.8 mg, 0.092 mmol), HATU (38 mg, 0.1 mmol) and triethylamine (17 mg, 0.168 mmol) to obtain compound 42g as light brown solid (16.7 mg, yield = 41%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.16 (br. s., 1H), 8.48 (d, J = 8.07 Hz, 1H), 7.52 (dd, J = 1.22, 8.07 Hz, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 7.18 (d, J = 7.58 Hz, 1H), 7.11 (d, J = 7.58 Hz, 1H), 6.98 (s, 1H), 4.19 (s, 3H), 3.07 (t, J = 5.26 Hz, 4H), 2.42 (s, 3H), 2.37 (s, 3H), 1.68 (quin, J = 5.50 Hz, 4H), 1.43 - 1.51 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 161.3, 157.7, 151.7, 141.9, 135.4, 133.6, 132.9, 130.8, 130.2, 129.0, 122.5, 120.2, 111.3, 110.8, 57.1, 46.9, 25.1, 23.4, 20.9, 20.5. HRMS for C₂₄H₂₈N₃O₄S₂ [M + H]⁺ calculated 486.1516, found 486.1507.

2-chloro-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (42h).

Compound 42h was synthesized using the same procedure as for 42d starting with 2-chloro-4-(chlorosulfonyl)benzoic acid (40h, 50 mg, 0.2 mmol) to obtain compound 41h (10.5 mg, yield = 18%). The ‘HATU coupling’ reaction was then performed using 41h (10.5 mg, 0.035 mmol), 8a (7.8 mg, 0.038 mmol), HATU (15.8 mg, 0.042 mmol) and triethylamine (7 mg, 0.069 mmol) to obtain compound 42h as white solid (12.3 mg, yield = 72%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.73 (s, 1H), 7.70 (d, J = 7.83 Hz, 1H), 7.58 (d, J = 8.07 Hz, 1H), 7.26 (s, 1H), 7.12 (d, J = 7.83 Hz, 1H), 7.06 (d, J = 7.58 Hz, 1H), 7.00 (s, 1H), 3.01 (t, J = 4.89 Hz, 4H), 2.35 (s, 3H), 2.34 (s, 3H), 1.67 (quin, J = 5.50 Hz, 4H), 1.44 - 1.49 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 162.8, 157.2, 140.2, 135.7, 135.6, 132.7, 132.6, 132.3, 131.2, 130.9, 129.8, 129.3, 129.2, 126.0, 111.3, 46.9, 25.1, 23.3, 21.0, 20.6. HRMS for C₂₃H₂₅ClN₃O₃S₂ [M + H]⁺ calculated 490.1020, found 490.1013.

2-bromo-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (42i).

Compound 42i was synthesized using the same procedure as for 42d starting with 2-bromo-4-(chlorosulfonyl)benzoic acid (40i, 50 mg, 0.2 mmol) to obtain compound 41i (7.7 mg, yield = 11%). The ‘HATU coupling’ reaction was then performed using 41i (7.7 mg, 0.022 mmol), 8a (4.9 mg, 0.024 mmol), HATU (10.1 mg, 0.027 mmol) and triethylamine (4.5 mg, 0.044 mmol) to obtain compound 42i as white solid (7.8 mg, yield = 66%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.82 (s, 1H), 7.49 (d, J = 8.07 Hz, 1H), 7.38 (d, J = 8.07 Hz, 1H), 7.19 (s, 1H), 7.10 (d, J = 7.83 Hz, 1H), 7.04 (d, J = 7.34 Hz, 1H), 6.98 (s, 1H), 2.96 (t, J = 4.77 Hz, 4H), 2.33 (s, 3H), 2.31 (s, 3H), 1.65 (quin, J = 5.50 Hz, 4H), 1.41 - 1.49 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.1, 157.7, 149.3, 139.5, 138.2, 135.6, 132.6, 132.4, 132.1, 131.3, 130.0, 129.6, 129.1, 126.3, 120.4, 111.4, 46.9, 25.0, 23.3, 21.1, 20.7. HRMS for C₂₃H₂₅BrN₃O₃S₂ [M + H]⁺ calculated 534.0515, found 534.0510.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-2-methoxy-4-(piperidin-1-ylsulfonyl)benzamide (42j).

Compound 42j was synthesized using the same procedure as for 42c starting with methyl 4-(chlorosulfonyl)-2-methoxybenzoate (40j, 50 mg, 0.19 mmol) to obtain compound 41j (48 mg, yield = 81%). The ‘HATU coupling’ reaction was then performed using 41j (21.5 mg, 0.072 mmol), 8a (19 mg, 0.079 mmol), HATU (38.5 mg, 0.086 mmol) and triethylamine (14.5 mg, 0.143 mmol) to obtain compound 42j as light yellow solid (22.8 mg, yield = 65%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.87 (d, J = 8.07 Hz, 1H), 7.58 (s, 1H), 7.35 (d, J = 8.07 Hz, 1H), 7.26 (s, 1H), 7.09 (d, J = 7.83 Hz, 1H), 6.95 - 7.04 (m, 2H), 3.97 (s, 3H), 3.21 (t, J = 5.10 Hz, 4H), 2.36 (s, 3H), 2.31 (s, 3H), 1.58 - 1.64 (m, 4H), 1.49 - 1.55 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 158.1, 157.0, 149.2, 136.7, 135.6, 132.9, 132.7, 131.9, 131.0, 130.9, 129.9, 129.2, 118.4, 111.7, 111.2, 56.2, 46.8, 25.7, 23.8, 20.9, 20.4. HRMS for C₂₄H₂₈N₃O₄S₂ [M + H]⁺ calculated 486.1516, found 486.1512.

3-amino-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (42k).

To a solution of compound 42f (19.7 mg, 0.039 mmol) in methanol, was added a catalytic amount of palladium on carbon and the flask was subjected to hydrogenation reaction using Parr shaker apparatus for 4h. The solvent was then filtered to obtain the filtrate which was concentrated under vacuum to obtain residue which was purified using C18 reverse phase column chromatography to compound 42k as white solid (10.5 mg, yield = 50%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.70 (d, J = 8.31 Hz, 1H), 7.55 (s, 1H), 7.39 - 7.44 (m, 1H), 7.18 (s, 2H), 6.96 (s, 1H), 3.14 (t, J = 5.40 Hz, 4H), 2.37 (br. s., 3H), 2.36 (br. s., 3H), 1.64 (quin, J = 5.60 Hz, 4H), 1.44 - 1.52 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.6, 153.1, 149.9, 146.5, 135.8, 132.9, 131.2, 131.1, 130.1, 129.8, 122.1, 117.2, 115.0, 110.7, 46.8, 25.1, 23.4, 20.9, 20.5. HRMS for C₂₃H₂₇N₄O₃S₂ [M + H]⁺ calculated 471.1519, found 471.1515.

4-(cyclohexylthio)-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)benzamide (45a).

To a solution of 4-mercaptobenzoic acid (43, 50 mg, 0.32 mmol) in anhydrous DMF were added, bromocyclohexane (56 mg, 0.34 mmol) and potassium carbonate (67 mg, 0.49 mmol). The reaction was stirred for 1h followed by removal of solvent under vacuum to obtain residue which was purified using silica gel column chromatography to yield compound 44 (15 mg, yield = 20%). The ‘HATU coupling’ reaction was then performed using 44 (13 mg, 0.055 mmol), 8a (13 mg, 0.061 mmol), HATU (23 mg, 0.061 mmol) and triethylamine (8 mg, 0.083 mmol) to obtain compound 45a as light yellow solid (9 mg, yield = 39%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.65 (br. s., 1H), 7.88 (d, J = 8.31 Hz, 2H), 7.37 (d, J = 8.31 Hz, 2H), 7.29 (d, J = 0.49 Hz, 1H), 7.12 - 7.16 (m, 1H), 7.06 - 7.10 (m, 1H), 6.94 (s, 1H), 3.28 - 3.40 (m, 1H), 2.37 (s, 3H), 2.34 (s, 3H), 2.02 - 2.11 (m, 2H), 1.82 (dd, J = 3.79, 8.93 Hz, 2H), 1.63 - 1.71 (m, 1H), 1.32 - 1.51 (m, 5H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.5, 159.0, 148.0, 143.9, 135.5, 132.9, 132.3, 130.8, 130.2, 129.4, 128.3, 128.1, 127.8, 110.7, 44.8, 33.0, 29.7, 25.6, 20.9, 20.3. HRMS for C₂₄H₂₇N₂OS₂ [M + H⁺] calculated 423.1559, found 423.1557.

4-(cyclohexylsulfonyl)-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)benzamide (45b).

To a solution of compounds 45a (7 mg, 0.017 mmol) in 4:1 mixture of (CH₂Cl₂/methanol was added 3-chloroperoxybenzoic acid (15 mg, 0.083 mmol). The reaction was heated at 45 °C for 30 minutes, followed by removal of solvent under vacuum to obtain residue which was purified using silica gel column chromatography to obtain compound 45b as off-white solid (5.5 mg, yield = 71%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.35 (d, J = 8.32 Hz, 2H), 8.05 (d, J = 8.56 Hz, 2H), 7.25 (s, 1H), 7.15 - 7.22 (m, 2H), 6.98 (s, 1H), 2.97 (tt, J = 3.39, 12.14 Hz, 1H), 2.36 (d, J = 2.20 Hz, 6H), 2.05 - 2.12 (m, 2H), 1.89 (d, J = 13.45 Hz, 2H), 1.69 (d, J = 12.23 Hz, 1H), 1.41 - 1.50 (m, 2H), 1.18 - 1.30 (m, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.4, 160.9, 145.2, 141.8, 135.9, 135.2, 133.2, 131.1, 130.6, 130.5, 129.7, 129.5, 129.1, 110.9, 63.5, 25.4, 25.0, 25.0, 20.8, 20.0. MS for C₂₄H₂₇N₂O₃S₂ [M + H⁺] calculated 455.1458, found 455.1456.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidine-1-carbonyl)benzamide (45c).

To a solution of terephthalic acid (46, 200 mg, 1.81 mmol) in anhydrous DMF were added, piperidine (3, 171 mg, 1.98 mmol), HATU (755 mg, 1.98 mmol) and triethylamine (364 mg, 3.61 mmol). The reaction was stirred for 1h followed by removal of the solvent. Water was then added to precipitate the HATU byproduct and the bis-amide side product which were removed by filtration. The aqueous layer was then acidified using 3N aqueous HCl to precipitate the solid which was collected by filtration to obtain compound 47 (98 mg, yield = 23%). The ‘HATU coupling’ reaction was then performed using 47 (26 mg, 0.11 mmol), 8a (25 mg, 0.12 mmol), HATU (46.6 mg, 0.12 mmol) and triethylamine (22.5 mg, 0.22 mmol) to obtain compound 45c as white solid (9.2 mg, yield = 20%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.45 (br. s., 1H), 7.89 (d, J = 8.07 Hz, 2H), 7.44 (d, J = 8.31 Hz, 2H), 7.33 (s, 1H), 7.10 (d, J = 7.58 Hz, 1H), 7.02 (d, J = 7.83 Hz, 1H), 6.98 (d, J = 0.49 Hz, 1H), 3.73 (br. s., 2H), 3.29 (br. s., 2H), 2.38 (s, 3H), 2.32 (s, 3H), 1.69 (br. s., 4H), 1.53 (br. s., 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 168.8, 164.0, 157.3, 150.2, 140.6, 135.3, 134.0, 132.7, 132.5, 130.8, 130.1, 128.8, 127.6, 127.2, 111.1, 48.6, 43.1, 26.5, 25.5, 24.5, 20.9, 20.5. HRMS for C₂₄H₂₆N₃O₂S [M⁺] calculated 420.1740, found 420.1738.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-3-(piperidine-1-carbonyl)benzamide (45d).

Compound 45d was synthesized using the same procedure as for 45c starting with isophthalic acid (48, 300mg, 1.81 mmol), piperidine (3, 171 mg, 1.98 mmol), HATU (755 mg, 1.98 mmol) and triethylamine (364 mg, 3.61 mmol) to obtain compound 49 which was purified by silica gel column chromatography. The ‘HATU coupling’ reaction was then performed using 49 (23 mg, 0.098 mmol), 8a (20 mg, 0.098 mmol), HATU (41 mg, 0.11 mmol) and triethylamine (20 mg, 0.196 mmol) to obtain compound 45d as off-white solid (12 mg, yield = 29%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.34 (br. s., 1H), 7.97 (s, 1H), 7.92 (d, J = 7.83 Hz, 1H), 7.62 (d, J = 7.58 Hz, 1H), 7.51 (t, J = 7.58 Hz, 1H), 7.35 (s, 1H), 7.12 (d, J = 7.83 Hz, 1H), 7.05 (d, J = 7.83 Hz, 1H), 6.98 (s, 1H), 3.74 (br. s., 2H), 3.34 (br. s., 2H), 2.39 (s, 3H), 2.34 (s, 3H), 1.70 (br. s., 4H), 1.55 (br. s., 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 168.8, 163.9, 157.2, 150.3, 137.2, 135.3, 133.9, 132.8, 132.1, 131.2, 130.8, 130.2, 129.2, 128.8, 128.1, 125.9, 111.1, 48.8, 43.3, 26.5, 25.5, 24.5, 20.9, 20.5. HRMS for C₂₄H₂₆N₃O₂S [M + H⁺] calculated 420.1740, found 420.1737.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperidine-1-carbonyl)benzenesulfonamide (51).

To a solution of 4-(chlorosulfonyl)phenyl acetate (2, 50 mg, 0.214 mmol) in anhydrous dichloromethane were added triethylamine (32 mg, 0.32 mmol) and compound 8a (44 mg, 0.321 mmol). The reaction mixture was heated at 45 °C for 16h followed by removal of the solvent to obtain the crude sulfonamide intermediate 4-(N-(4-(2,5-dimethylphenyl)thiazol-2-yl)sulfamoyl)benzoic acid which was suspended in water and filtered to remove the water soluble impurities to obtain the methyl ester intermediate (21 mg, yield = 24%). This intermediate (21 mg, 0.052 mmol) was dissolved in 3:1 mixture of MeOH/THF and LiOH (4 mg, 0.156 mmol) dissolved in 0.2 mL of water was added to this solution. The reaction was stirred for another 2h followed by removal of the solvent under vacuum. The residue was then dissolved in water and the solution was acidified using 3M HCl to precipitate the product which was purified by filtration to obtain compound 50 (13 mg, yield = 64%). The ‘HATU coupling’ reaction was then performed using 50 (13 mg, 0.034 mmol), piperidine (3, 2.84 mg, 0.034 mmol), HATU (14 mg, 0.037 mmol) and triethylamine (5.1 mg, 0.05 mmol) to obtain compound 51 as tan solid (15 mg, yield = 97%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.97 (d, J = 8.07 Hz, 2H), 7.46 (d, J = 8.07 Hz, 2H), 7.17 (s, 2H), 7.10 (s, 1H), 6.29 (s, 1H), 3.66 (br. t, J = 5.10 Hz, 2H), 3.29 (t, J = 5.10 Hz, 2H), 2.34 (s, 3H), 2.32 (s, 3H), 1.63 - 1.70 (m, 4H), 1.51 (br. s.,2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 168.9, 168.8, 142.8, 139.8, 136.3, 136.0, 133.2, 131.0, 130.6, 129.7, 128.3, 127.1, 126.7, 103.9, 48.7, 43.2, 26.4, 25.5, 24.4, 20.8, 19.9. HRMS for C₂₃H₂₆N₃O₃S [M + H⁺] calculated 456.1410, found 456.1411

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(N-ethyl-N-propylsulfamoyl)benzamide (54a).

To a solution of 4-(chlorosulfonyl)benzoic acid (52, 100 mg, 0.45 mmol) in anhydrous DMF were added, potassium carbonate (68 mg, 0.495 mmol) and N-ethylpropan-1-amine (39 mg, 0.45 mmol). The reaction was heated at 45 °C for 30 minutes followed by removal of solvent under vacuum. Water as then added and the solution was acidified with 3N HCl to precipitate the solid which was collected by filtration to obtain crude compound 53a. The ‘HATU coupling’ reaction was then performed using 53a (23 mg, 0.083 mmol), 8a (17 mg, 0.083 mmol), HATU (35 mg, 0.091 mmol) and triethylamine (12.6 mg, 0.13 mmol) to obtain compound 54a as glassy oil (27 mg, yield = 71%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.19 (br. s., 1H), 7.78 (d, J = 8.31 Hz, 2H), 7.69 (d, J = 8.31 Hz, 2H), 7.13 (s, 1H), 6.94 - 7.01 (m, 2H), 6.87 - 6.91 (m, 1H), 3.20 (q, J = 7.17 Hz, 2H), 3.08 (dd, J = 6.97, 8.19 Hz, 2H), 2.29 (s, 3H), 2.24 (s, 3H), 1.55 (qd, J = 7.40, 14.98 Hz, 2H), 1.08 (t, J = 7.09 Hz, 3H), 0.88 (t, J = 7.34 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.0, 158.5, 150.0, 143.6, 135.4, 135.0, 133.3, 132.5, 130.9, 129.9, 129.0, 128.1, 127.0, 111.3, 49.2, 42.6, 21.9, 20.8, 20.3, 14.0, 11.1. HRMS for C₂₃H₂₈N₃O₃S₂ [M + H]⁺ calculated 458.1567, found 458.1562.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(N,N-dipropylsulfamoyl)benzamide (54b).

Compound 54b was synthesized using the same procedure as for 54a starting with compound 52 (100 mg, 0.45 mmol), potassium carbonate (68 mg, 0.495 mmol) and dipropylamine (50 mg, 0.5 mmol) to obtain compound 53b. The ‘HATU coupling’ reaction was then performed using 53b (17.5 mg, 0.06 mmol), 8a (12.5 mg, 0.06 mmol), HATU (26 mg, 0.067 mmol) and triethylamine (9 mg, 0.09 mmol) to obtain compound 54b as glassy oil (19 mg, yield = 67%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.26 (br. s., 1H), 7.75 - 7.82 (m, 2H), 7.65 - 7.72 (m, 2H), 7.13 (s, 1H), 6.99 (s, 1H), 6.97 (d, J = 7.83 Hz, 1H), 6.87 - 6.91 (m, 1H), 3.03 - 3.08 (m, 4H), 2.29 (s, 3H), 2.24 (s, 3H), 1.48 - 1.56 (m, 4H), 0.85 (t, J = 7.34 Hz, 6H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.1, 158.5, 150.0, 143.5, 135.4, 134.9, 133.3, 132.5, 130.9, 129.9, 129.0, 128.1, 127.0, 111.3, 49.9, 21.9, 20.8, 20.2, 11.1. HRMS for C₂₄H₃₀N₃O₃S₂ [M + H⁺] calculated 472.1723, found 472.1717.

4-(N,N-dibutylsulfamoyl)-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)benzamide (54c).

Compound 54c was synthesized using the same procedure as for 54a starting with compound 52 (100 mg, 0.45 mmol), potassium carbonate (68 mg, 0.497 mmol) and dibutylamine (64 mg, 0.497 mmol) to obtain compound 53c. The ‘HATU coupling’ reaction was then performed using 53c (20 mg, 0.064 mmol), 8a (13 mg, 0.064 mmol), HATU (27 mg, 0.071 mmol) and triethylamine (9.7 mg, 0.096 mmol) to obtain compound 54c as off-white solid (16 mg, yield = 50%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.14 (br. s., 1H), 7.78 (d, J = 8.31 Hz, 2H), 7.68 (d, J = 8.56 Hz, 2H), 7.14 (s, 1H), 6.96 - 6.99 (m, 2H), 6.89 (d, J = 7.83 Hz, 1H), 3.07 - 3.12 (m, 4H), 2.29 (s, 3H), 2.24 (s, 3H), 1.43 - 1.51 (m, 4H), 1.24 - 1.31 (m, 4H), 0.89 (t, J = 7.46 Hz, 6H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.0, 158.5, 150.0, 143.5, 135.4, 135.0, 133.3, 132.5, 130.9, 129.9, 129.0, 128.1, 127.0, 111.3, 47.9, 30.6, 20.8, 20.3, 19.9, 13.7. HRMS for C₂₆H₃₄N₃O₃S₂ [M + H]⁺ calculated 500.2036, found 500.2029.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(N-ethyl-N-phenylsulfamoyl)benzamide (54d).

To a solution of methyl 4-(chlorosulfonyl)benzoate (2, 50 mg, 0.214 mmol) in anhydrous CH₂Cl₂ were added, triethylamine (32 mg, 0.321 mmol) and N-ethylaniline (26 mg, 0.214 mmol). The reaction was stirred for 30 minutes followed by removal of solvent under vacuum. Water as then added to precipitate the solid which was collected by filtration to obtain the methyl ester intermediate. This intermediate was then dissolved in 3:1 MeOH/THF and an aqueous solution of LiOH (27 mg, 0.642 mmol) was added. The reaction was stirred for 4h followed by removal of solvent under vacuum. Water was then added and the solution was acidified with 3N HCl to precipitate the solid which was collected by filtration to obtain crude compound 53d. The ‘HATU coupling’ reaction was then performed using 53d (20 mg, 0.066 mmol), 8a (13.5 mg, 0.066 mmol), HATU (27 mg, 0.072 mmol) and triethylamine (10 mg, 0.098 mmol) to obtain compound 54d as white solid (26 mg, yield = 80%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 12.61 (br. s., 1H), 7.66 (d, J = 8.31 Hz, 2H), 7.42 (d, J = 8.31 Hz, 2H), 7.27 - 7.32 (m, 3H), 7.04 (s, 1H), 6.94 - 6.99 (m, 3H), 6.91 (d, J = 7.83 Hz, 1H), 6.83 (dd, J = 1.10, 7.70 Hz, 1H), 3.56 (q, J = 7.17 Hz, 2H), 2.23 (s, 3H), 2.18 (s, 3H), 1.07 (t, J = 7.09 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.3, 158.8, 153.6, 150.0, 141.4, 138.1, 135.4, 135.3, 133.1, 132.4, 130.9, 129.8, 129.1, 129.0, 128.8, 128.1, 127.8, 127.5, 111.3, 45.7, 20.7, 20.2, 14.0. HRMS for C₂₆H₂₆N₃O₃S₂ [M + H]⁺ calculated 492.1410, found 492.1404.

4-(N-cyclohexylsulfamoyl)-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)benzamide (54e).

Compound 54e was synthesized using the same procedure as for 54a starting with compound 52 (100 mg, 0.45 mmol), potassium carbonate (68 mg, 0.497 mmol) and cyclohexylamine (49 mg, 0.497 mmol) to obtain compound 53e. The ‘HATU coupling’ reaction was then performed using 53e (13.5 mg, 0.048 mmol), 8a (9.7 mg, 0.048 mmol), HATU (20 mg, 0.052 mmol) and triethylamine (7.2 mg, 0.072 mmol) to obtain compound 54e as off-white solid (6.6 mg, yield = 29%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.46 (br. s., 1H), 7.92 - 7.96 (m, 2H), 7.84 - 7.88 (m, 2H), 7.22 (s, 1H), 7.05 (d, J = 7.83 Hz, 1H), 6.96 - 7.00 (m, 2H), 5.04 (d, J = 7.83 Hz, 1H), 3.07 - 3.17 (m, 1H), 2.33 (s, 3H), 2.28 (s, 3H), 1.67 (dd, J = 3.06, 12.59 Hz, 2H), 1.55 - 1.61 (m, 2H), 1.45 - 1.52 (m, 1H), 1.13 - 1.22 (m, 2H), 0.93 - 1.05 (m, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 158.1, 150.0, 145.2, 135.4, 135.3, 133.7, 132.8, 130.9, 130.1, 129.1, 128.3, 127.0, 111.4, 52.9, 33.8, 24.9, 24.7, 20.9, 20.3. HRMS for C₂₄H₂₈N₃O₃S₂ [M + H⁺] calculated 470.1567, found 470.1562.

tert-butyl 4-((4-((4-(2,5-dimethylphenyl)thiazol-2-yl)carbamoyl)phenyl)sulfonyl)piperazine-1-carboxylate (54f).

Compound 54f was synthesized using the same procedure as for 54d starting with compound 2 (100 mg, 0.43 mmol), triethylamine (65 mg, 0.65 mmol), tert-butyl piperazine-1-carboxylate (87 mg, 0.47 mmol) and LiOH (31 mg, 1.29 mmol) to obtain compound 53f. The ‘HATU coupling’ reaction was then performed using 53f (75 mg, 0.203 mmol), 8a (41 mg, 0.203 mmol), HATU (85 mg, 0.223 mmol) and triethylamine (31 mg, 0.31 mmol) to obtain compound 54f as white solid (109 mg, yield = 96%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.90 (br. s., 1H), 7.87 (d, J = 8.56 Hz, 2H), 7.66 (d, J = 8.31 Hz, 2H), 7.17 (s, 1H), 6.97 - 7.04 (m, 2H), 6.92 (s, 1H), 3.51 (t, J = 4.52 Hz, 4H), 2.96 (br. s., 4H), 2.31 (s, 3H), 2.25 (s, 3H), 1.40 (s, 9H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 158.2, 154.0, 152.9, 149.9, 135.8, 135.4, 133.3, 132.5, 131.0, 129.9, 129.0, 128.2, 127.8, 111.5, 80.5, 76.4, 45.8, 28.3, 20.8, 20.3. HRMS for C₂₇H₃₃N₄O₅S₂ [M + H]⁺ calculated 557.1887, found 557.1886.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-ethoxypiperidin-1-yl)sulfonyl)benzamide (54g).

Compound 54g was synthesized using the same procedure as for 54d starting with compound 2 (100 mg, 0.43 mmol), triethylamine (130 mg, 1.29 mmol), 4-ethoxypiperidine hydrochloride (78 mg, 0.47 mmol) and LiOH (31 mg, 1.29 mmol) to obtain compound 53g. The ‘HATU coupling’ reaction was then performed using 53g (25 mg, 0.08 mmol), 8a (16 mg, 0.08 mmol), HATU (37 mg, 0.097 mmol) and triethylamine (16 mg, 0.16 mmol) to obtain compound 54g as off-white solid (35 mg, yield = 88%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.97 (br. s., 1H), 7.85 (d, J = 8.31 Hz, 2H), 7.67 (d, J = 8.31 Hz, 2H), 7.16 (s, 1H), 6.98 - 7.02 (m, 2H), 6.90 - 6.94 (m, 1H), 3.40 (q, J = 6.93 Hz, 2H), 3.33 - 3.37 (m, 1H), 3.14 - 3.21 (m, 2H), 2.94 (ddd, J = 3.42, 7.40, 11.19 Hz, 2H), 2.31 (s, 3H), 2.25 (s, 3H), 1.83 - 1.90 (m, 2H), 1.68 - 1.73 (m, 2H), 1.11 (t, J = 7.09 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.9, 158.3, 150.0, 139.9, 135.4, 133.4, 132.5, 130.9, 129.9, 129.0, 128.1, 127.7, 111.4, 72.0, 63.4, 43.1, 30.2, 20.8, 20.3, 15.4. HRMS for C₂₅H₃₀N₃O₄S₂ [M + H]⁺ calculated 500.1672, found 500.1666.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide (54h).

Compound 54h was synthesized using the same procedure as for 54d starting with compound 2 (100 mg, 0.43 mmol), triethylamine (87 mg, 0.86 mmol), 4-propylperidine (60 mg, 0.47 mmol) and LiOH (31 mg, 1.29 mmol) to obtain compound 537h. The ‘HATU coupling’ reaction was then performed using 53h (25 mg, 0.08 mmol), 8a (16 mg, 0.08 mmol), HATU (37 mg, 0.097 mmol) and triethylamine (16 mg, 0.16 mmol) to obtain compound 54g as off-white solid (33 mg, yield = 82%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.68 (br. s., 1H), 7.86 (d, J = 8.31 Hz, 2H), 7.69 (d, J = 8.31 Hz, 2H), 7.19 (s, 1H), 6.99 - 7.03 (m, 2H), 6.94 (d, J = 7.58 Hz, 1H), 3.76 (d, J = 11.49 Hz, 2H), 2.32 (s, 3H), 2.27 (s, 3H), 2.17 - 2.24 (m, 2H), 1.71 (d, J = 11.98 Hz, 2H), 1.22 - 1.30 (m, 4H), 1.16 - 1.21 (m, 3H), 0.85 (t, J = 7.21 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.8, 150.1, 139.9, 135.4, 135.4, 133.4, 132.5, 130.9, 129.9, 129.0, 128.0, 127.8, 111.4, 46.5, 38.2, 34.7, 31.4, 20.8, 20.3, 19.6, 14.1. HRMS for C₂₆H₃₂N₃O₃S₂ [M + H]⁺ calculated 498.1880, found 498.1879.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(piperazin-1-ylsulfonyl)benzamide (54i).

Compound 54f (105 mg, 0.189 mmol) was stirred in 4N HCl solution in dioxane for 30 minutes. The solvent was then removed to obtain the hydrochloride salt of compound 54i as tan solid in quantitative yield. ¹H NMR (500 MHz, METHANOL-d₄) δ 8.30 (d, J = 8.56 Hz, 2H), 8.02 (d, J = 8.56 Hz, 2H), 7.40 (s, 1H), 7.15 - 7.18 (m, 2H), 7.10 (dd, J = 1.47, 7.83 Hz, 1H), 3.33 - 3.38 (m, 8H), 2.40 (s, 3H), 2.34 (s, 3H). ¹³C NMR (126 MHz, METHANOL-d₄) δ 166.5, 151.1, 140.1, 138.9, 136.6, 135.4, 135.4, 134.4, 131.9, 131.4, 130.5, 130.1, 129.5, 112.5, 68.3, 44.4, 21.1, 20.9. HRMS for C₂₂H₂₅N₄O₃S₂ [M + H]⁺ calculated 457.1363, found 457.1365.

4-((4-acetylpiperazin-1-yl)sulfonyl)-N-(4-(2,5-dimethylphenyl)thiazol-2-yl)benzamide (54j).

To a solution of compound 54i (20 mg, 0.041 mmol) in anhydrous THF were added, triethylamine (10.4 mg, 0.1 mmol) and acetyl chloride (4 mg, 0.049 mmol). The reaction was stirred for 1h followed by removal of solvent under vacuum to obtain residue which was purified using silica gel column chromatography to obtain compound 54j as tan solid (16 mg, yield = 78%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.83 (br. s., 1H), 7.90 (d, J = 8.07 Hz, 2H), 7.68 (d, J = 8.07 Hz, 2H), 7.17 (s, 1H), 6.98 - 7.02 (m, 2H), 6.93 (d, J = 7.83 Hz, 1H), 3.70 (br. s., 2H), 3.55 (t, J = 4.40 Hz, 2H), 2.94 - 3.04 (m, 4H), 2.31 (s, 3H), 2.26 (s, 3H), 2.03 (s, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 168.8, 163.6, 158.2, 154.0, 152.9, 150.0, 138.7, 136.0, 135.4, 133.4, 132.5, 130.9, 129.9, 129.0, 128.3, 127.8, 111.5, 46.0, 45.7, 45.6, 40.6, 21.2, 20.8, 20.3. HRMS for C₂₄H₂₇N₄O₄S₂ [M + H]⁺ calculated 499.1468, found 499.1473.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-propylpiperazin-1-yl)sulfonyl)benzamide (54k).

To a solution of compound 54i (20 mg, 0.041 mmol) in anhydrous THF were added, triethylamine (10.4 mg, 0.1 mmol) and 1-iodopropane (7.7 mg, 0.045 mmol). The reaction was stirred for 4h followed by removal of solvent under vacuum to obtain residue which was purified using silica gel column chromatography to obtain compound 54k as tan solid (5 mg, yield = 25%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.84 (br. s., 1H), 7.87 (d, J = 8.56 Hz, 2H), 7.67 (d, J = 8.56 Hz, 2H), 7.16 (s, 1H), 6.96 - 7.01 (m, 2H), 6.91 (dd, J = 1.10, 7.70 Hz, 1H), 3.06 (br. s., 4H), 2.55 (br. s., 4H), 2.29 - 2.35 (m, 5H), 2.25 (s, 3H), 1.41 - 1.50 (m, 2H), 0.86 (t, J = 7.46 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 158.3, 150.0, 138.8, 135.6, 135.4, 133.4, 132.5, 130.9, 129.9, 129.0, 128.1, 127.9, 111.4, 60.0, 52.0, 45.7, 20.8, 20.3, 19.7, 11.7. HRMS for C₂₅H₃₁N₄O₃S₂ [M + H]⁺ calculated 499.1832, found 499.1834.

N-(4-(2-bromo-5-methylphenyl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide (55).

The ‘HATU coupling’ reaction was performed using 53h (17 mg, 0.055 mmol), 8d (15 mg, 0.055 mmol), HATU (25 mg, 0.066 mmol) and triethylamine (11.3 mg, 0.11 mmol) to obtain compound 55 as oil (9.1 mg, yield = 30%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.93 - 8.12 (m, J = 8.32 Hz, 2H), 7.73 - 7.82 (m, J = 8.31 Hz, 2H), 7.40 (d, J = 8.31 Hz, 1H), 7.24 (s, 1H), 7.20 (s, 1H), 6.89 - 6.98 (m, 1H), 3.73 (d, J = 11.74 Hz, 2H), 2.24 (s, 3H), 2.20 (t, J = 11.13 Hz, 2H), 1.66 (d, J = 12.23 Hz, 2H), 1.15 - 1.27 (m, 4H), 1.07 - 1.15 (m, 3H), 0.78 (t, J = 7.21 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.1, 159.8, 145.5, 140.6, 137.8, 134.5, 133.5, 132.2, 131.9, 131.3, 128.7, 128.0, 118.7, 113.0, 46.5, 38.2, 34.7, 31.4, 20.8, 19.6, 14.1. HRMS for C₂₅H₂₉BrN₃O₃S₂ [M + H]⁺ calculated 562.0828 found 562.0829.

N-(4-(2,5-dimethylphenyl)-5-(pent-1-yn-1-yl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide (57).

The ‘HATU coupling’ reaction was performed using 53h (12 mg, 0.039 mmol), 25 (10.9 mg, 0.039 mmol), HATU (17.5 mg, 0.046 mmol) and triethylamine (7.8 mg, 0.077 mmol) to obtain compound 56 (12 mg, yield = 53%). Sonogashira coupling reaction was performed using copper iodide (0.2 mg, 0.001 mmol), tetrakis(triphenylphosphine) palladium(0) (2.41 mg, 0.002 mmol), 1-pentyne (4.2 mg, 0.06 mmol), diisopropylamine (0.45 mL) and compound 56 (12 mg, 0.021 mmol) to obtain compound 57 as tan solid (4.9 mg, yield = 41%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.99 - 8.11 (m, 2H), 7.75 - 7.91 (m, J = 8.31 Hz, 2H), 7.21 (s, 1H), 7.03 - 7.13 (m, 2H), 3.80 (d, J = 11.49 Hz, 2H), 2.35 (t, J = 6.80 Hz, 2H), 2.32 (s, 3H), 2.22 - 2.29 (m, 5H), 1.73 (d, J = 12.72 Hz, 2H), 1.55 (sxt, J = 7.10 Hz, 2H), 1.25 - 29 (m, 5H), 1.16 - 1.22 (m, 3H), 0.95 (t, J = 7.34 Hz, 2H), 0.86 (t, J = 7.09 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.9, 157.0, 154.0, 140.7, 135.1, 134.5, 133.9, 132.6, 130.7, 130.6, 130.2, 128.7, 128.1, 110.9, 99.8, 70.3, 46.5, 38.2, 34.7, 31.4, 21.8, 21.7, 20.8, 19.6, 19.6, 14.1, 13.4. HRMS for C₃₁H₃₈N₃O₃S₂ [M + H]⁺ calculated 564.2349, found 564.2349.

N-(4-(2-bromo-5-methylphenyl)-5-(pent-1-yn-1-yl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (60).

To a solution of compound 8d (144 mg, 0.54 mmol) in anhydrous dichloromethane was slowly added a solution of N-iodosuccinimide (144 mg, 0.64 mmol) in 5 mL of dichloromethane and the reaction was stirred for 30 minutes. The solvent was then removed and the residue was purified using column chromatography to obtain compound 58 (160 mg, yield = 76%). Sonogashira coupling reaction was then performed using copper iodide (1.2 mg, 0.006 mmol), tetrakis(triphenylphosphine)palladium(0) (14.7 mg, 0.02 mmol), 1-pentyne (43 mg, 0.63 mmol), diisopropylamine (0.45 mL) and compound 58 (50 mg, 0.13 mmol) to obtain compound 59 (7 mg, yield = 16%). The ‘HATU coupling’ reaction was then performed using 4 (4.2 mg, 0.023 mmol), 59 (7 mg, 0.021 mmol), HATU (9.5 mg, 0.025 mmol) and triethylamine (16 mg, 0.42 mmol) to obtain compound 60 as tan solid (3.1 mg, yield = 25%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.96 (d, J = 8.31 Hz, 2H), 7.78 (d, J = 8.31 Hz, 2H), 7.39 (d, J = 8.07 Hz, 1H), 7.22 (s, 1H), 6.92 (d, J = 7.83 Hz, 1H), 2.99 (t, J = 4.90 Hz, 4H), 2.35 (t, J = 6.97 Hz, 2H), 2.28 (s, 3H), 1.63 - 1.67 (m, 5H), 1.54 (sxt, J = 7.30 Hz, 3H), 1.40 - 1.46 (m, 2H), 0.93 (t, J = 7.34 Hz, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.6, 155.7, 150.6, 140.1, 137.1, 134.9, 133.9, 133.0, 132.5, 130.9, 128.2, 127.9, 119.2, 112.4, 99.3, 70.7, 46.9, 25.1, 23.4, 21.8, 21.7, 20.8, 13.3. HRMS for C₂₇H₂₉BrN₃O₃S₂ [M + H]⁺ calculated 586.0828, found 586.0820.

N-(4-(2-bromo-5-methylphenyl)-5-(pent-1-yn-1-yl)thiazol-2-yl)-4-((4-propylpiperidin-1-yl)sulfonyl)benzamide (61).

The ‘HATU coupling’ reaction was then performed using 53h (7.6 mg, 0.024 mmol), 59 (7.4 mg, 0.022 mmol), HATU (10.1 mg, 0.026 mmol) and triethylamine (4.5 mg, 0.045 mmol) to obtain compound 61 as yellow solid (6.4 mg, yield = 46%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 8.01 (d, J = 8.07 Hz, 2H), 7.79 (d, J = 8.31 Hz, 2H), 7.41 (d, J = 8.07 Hz, 1H), 7.23 (s, 1H), 6.95 (d, J = 7.34 Hz, 1H), 3.78 (d, J = 11.25 Hz, 2H), 2.35 (t, J = 6.97 Hz, 2H), 2.29 (s, 3H), 2.23 (t, J = 11.00 Hz, 2H), 1.72 (d, J = 12.23 Hz, 2H), 1.54 (sxt, J = 7.30 Hz, 2H), 1.24 - 1.28 (m, 4H), 1.16 - 1.21 (m, 3H), 0.89 - 0.97 (m, 3H), 0.85 (t, J = 7.09 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 156.2, 149.6, 140.3, 137.1, 134.8, 133.3, 133.0, 132.5, 131.2, 128.4, 128.0, 119.4, 112.3, 99.7, 70.4, 46.5, 38.2, 34.7, 31.4, 21.8, 21.7, 20.8, 19.6, 14.1, 13.3. HRMS for C₃₀H₃₅BrN₃O₃S₂ [M + H]⁺ calculated 628.1298, found 628.1300.

N-(4-(5-azido-2-methylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (63).

To a Compound 8e (50 mg, 0.186 mmol), sodium azide (49 mg, 0.746 mmol) and copper iodide (106 mg, 0.558 mmol) were taken in a microwave vial, sealed and evacuated under vacuum. Separately, 1,2-dimethylethylenediamine (66 mg, 0.744 mmol) was dissolved in DMF (1.2 mL) and was added to the vial. The reaction was heated in microwave at 100 °C for 15 min. Solvent was then removed and the residue was dissolved in EtOAc, washed with water and brine, dried over sodium sulfate and evaporated under vacuum to obtain crude compound which was purified using silica gel column chromatography to obtain compound 62 (9 mg, yield = 21%). The ‘HATU coupling’ reaction was then performed using 4 (10 mg, 0.037 mmol), 62 (8.6 mg, 0.037 mmol), HATU (15.4 mg, 0.041 mmol) and triethylamine (5.6 mg, 0.056 mmol) to obtain compound 63 as orange solid (10 mg, yield = 56%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.08 - 11.50 (m, 1H), 7.94 (s, 2H), 7.76 (d, J = 8.31 Hz, 2H), 7.11 - 7.16 (m, 2H), 7.06 (s, 1H), 6.83 (dd, J = 2.45, 8.07 Hz, 1H), 3.00 (br. s., 4H), 2.37 (s, 3H), 1.65 (br. s., 4H), 1.40 - 1.46 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.9, 158.3, 149.0, 140.5, 137.9, 135.6, 135.3, 132.7, 132.6, 128.3, 128.1, 119.8, 118.9, 112.5, 47.1, 25.3, 23.6, 20.8 MS for C₂₂H₂₃N₆O₃S₂ [M + H]⁺ calculated 483.1268, found 483.1263.

N-(4-(2-azido-5-methylphenyl)thiazol-2-yl)-4-(piperidin-1-ylsulfonyl)benzamide (66).

To a solution of 1-(2-azido-5-methylphenyl)ethan-1-one (64, 17 mg, 0.097 mmol) in chloroform was added liquid bromine (17 mg, 0.11 mmol). The reaction was stirred for an hour, followed by removal of the solvent to obtain crude brominated intermediate which was dissolved in anhydrous EtOH and thiourea (15 mg, 0.194 mmol) was added. The reaction was then heated in microwave at 100 °C for 30 min, followed by removal of the solvent to obtain the residue which was purified using silica gel column chromatography to obtain compound 65 (11 mg, yield = 49%). The ‘HATU coupling’ reaction was then performed using 4 (13 mg, 0.047 mmol), 65 (11 mg, 0.047 mmol), HATU (20 mg, 0.052 mmol) and triethylamine (7 mg, 0.071 mmol) to obtain compound 66 as light yellow solid (9 mg, yield = 40%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.02 - 11.59 (m, 1H), 7.99 (d, J = 8.31 Hz, 2H), 7.76 (d, J = 8.31 Hz, 2H), 7.62 (s, 1H), 7.57 (s, 1H), 7.07 (s, 2H), 2.94 - 3.01 (m, 4H), 2.28 (s, 3H), 1.65 (dd, J = 5.75, 11.37 Hz, 4H), 1.40 - 1.46 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 164.0, 154.3, 145.7, 140.4, 135.7, 134.8, 134.3, 130.6, 130.1, 128.3, 128.2, 125.2, 119.3, 113.5, 47.1, 25.3, 23.6, 21.1 MS for C₂₂H₂₃N₆O₃S₂ [M + H]⁺ calculated 483.1268, found 483.1262.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(N-(prop-2-yn-1-yl)sulfamoyl)benzamide (68a).

Compound 68a was synthesized using the same procedure as 54a starting with compound 52 (50 mg, 0.23 mmol), potassium carbonate (32 mg, 0.23 mmol) and propargylamine hydrochloride (23 mg, 0.25 mmol) separately treated with triethylamine (27 mg, 0.27mmol) to obtain compound 67a (26 mg, yield = 47%). The ‘HATU coupling’ reaction was then performed using 67a (22 mg, 0.09 mmol), 8a (19 mg, 0.09 mmol), HATU (38 mg, 0.101 mmol) and triethylamine (14 mg, 0.135 mmol) to obtain compound 68a as off-white solid (12 mg, yield = 31%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.13 - 11.51 (m, 1H), 8.06 - 8.11 (m, 2H), 7.95 - 8.01 (m, 2H), 7.10 - 7.14 (m, 1H), 7.06 (dd, J = 1.22, 7.58 Hz, 1H), 6.98 (s, 1H), 5.09 - 5.24 (m, 1H), 3.85 (br. s., 2H), 2.35 (s, 3H), 2.32 (s, 3H), 2.09 (s, 1H), 2.06 (t, J = 2.45 Hz, 1H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 154.0, 154.0, 143.5, 135.8, 135.4, 133.5, 132.8, 130.8, 130.1, 129.1, 128.3, 127.8, 111.3, 77.5, 73.3, 32.8, 20.9, 20.3. MS for C₂₁H₂₀N₃O₃S₂ [M + H]⁺ calculated 426.0941, found 426.0937.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(N-ethyl-N-(prop-2-yn-1-yl)sulfamoyl)benzamide (68b).

Compound 68b was synthesized using the same procedure as 54d starting with compound 2 (100 mg, 0.43 mmol), triethylamine (109 mg, 1.08 mmol), N-ethylprop-2-yn-1-amine hydrochloride (51 mg, 0.43 mmol) and LiOH (40 mg, 0.96 mmol) to obtain compound 67b (82 mg, yield = 71%). The ‘HATU coupling’ reaction was then performed using 67b (23 mg, 0.086 mmol), 8a (17.6 mg, 0.086 mmol), HATU (36 mg, 0.095 mmol) and triethylamine (17 mg, 0.17 mmol) to obtain compound 68b as off-white solid (32 mg, yield = 82%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.71 (br. s., 1H), 7.81 - 7.85 (m, 2H), 7.76 - 7.80 (m, 2H), 7.18 (s, 1H), 6.99 - 7.02 (m, 2H), 6.93 (dd, J = 1.35, 7.70 Hz, 1H), 4.16 (d, J = 2.45 Hz, 2H), 3.28 (q, J = 7.09 Hz, 2H), 2.32 (s, 3H), 2.27 (s, 3H), 1.96 (t, J = 2.45 Hz, 1H), 1.20 (t, J = 7.21 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.8, 158.1, 150.1, 142.4, 135.4, 135.4, 133.4, 132.6, 130.9, 129.9, 129.0, 127.9, 127.8, 111.4, 76.0, 73.9, 41.3, 35.6, 20.8, 20.3, 13.1. MS for C₂₃H₂₄N₃O₃S₂ [M + H]⁺ calculated 454.1254, found 454.1250.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-(N-(prop-2-yn-1-yl)-N-propylsulfamoyl)benzamide (68c).

Compound 68c was synthesized using the same procedure as 54d starting with compound 2 (90 mg, 0.39 mmol), triethylamine (99 mg, 0.98 mmol), N-proylprop-2-yn-1-amine hydrochloride (52 mg, 0.39 mmol) and LiOH (40 mg, 0.96 mmol) to obtain compound 67c (78 mg, yield = 71%). The ‘HATU coupling’ reaction was then performed using 67c (20 mg, 0.071 mmol), 8a (14.5 mg, 0.071 mmol), HATU (30 mg, 0.078 mmol) and triethylamine (11 mg, 0.11 mmol) to obtain compound 68c as off-white solid (25 mg, yield = 75%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.64 (br. s., 1H), 7.83 - 7.87 (m, 2H), 7.78 - 7.81 (m, 2H), 7.19 (s, 1H), 7.00 - 7.04 (m, 1H), 7.00 (s, 1H), 6.92 - 6.97 (m, 1H), 4.15 (d, J = 2.45 Hz, 2H), 3.16 (dd, J = 6.85, 7.83 Hz, 2H), 2.32 (s, 3H), 2.27 (s, 3H), 1.94 (t, J = 2.45 Hz, 1H), 1.56 - 1.64 (m, 2H), 0.94 (t, J = 7.46 Hz, 3H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 154.0, 150.1, 142.4, 135.4, 135.4, 134.0, 133.4, 132.6, 130.9, 129.9, 129.0, 127.9, 127.9, 111.3, 76.7, 75.9, 74.0, 47.9, 36.0, 20.8, 20.7, 20.3, 11.0. MS for C₂₄H₂₆N₃O₃S₂ [M + H]₊ calculated 468.1410, found 468.1408.

N-(4-(2,5-dimethylphenyl)thiazol-2-yl)-4-((4-ethynylpiperidin-1-yl)sulfonyl)benzamide (68d).

Compound 68d was synthesized using the same procedure as 54d starting with compound 2 (90 mg, 0.39 mmol), triethylamine (117 mg, 1.15 mmol), 4-ethynylpiperidine hydrochloride (71 mg, 0.488 mmol) and LiOH (36 mg, 0.86 mmol) to obtain compound 67d (72 mg, yield = 63%). The ‘HATU coupling’ reaction was then performed using 67d (25 mg, 0.085 mmol), 8a (17.5 mg, 0.085 mmol), HATU (36 mg, 0.094 mmol) and triethylamine (17 mg, 0.17 mmol) to obtain compound 68d as white solid (27.5 mg, yield = 67%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.81 (br. s., 1H), 7.86 (d, J = 8.56 Hz, 2H), 7.69 (d, J = 8.56 Hz, 2H), 7.18 (s, 1H), 6.99 - 7.03 (m, 2H), 6.93 (d, J = 7.83 Hz, 1H), 3.18 - 3.25 (m, 2H), 2.95 (t, J = 7.83 Hz, 2H), 2.51 (dt, J = 3.06, 6.79 Hz, 1H), 2.32 (s, 3H), 2.26 (s, 3H), 2.03 (d, J = 2.45 Hz, 1H), 1.86 - 1.94 (m, 2H), 1.70 - 1.79 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.8, 158.2, 150.1, 139.8, 135.5, 135.4, 133.4, 132.5, 130.9, 129.9, 129.0, 128.1, 127.7, 111.4, 85.0, 70.5, 44.0, 30.4, 25.5, 20.8, 20.3. MS for C₂₅H₂₆N₃O₃S₂ [M + H]⁺ calculated 480.1410, found 480.1409.

N-(4-(5-azido-2-methylphenyl)thiazol-2-yl)-4-((4-ethynylpiperidin-1-yl)sulfonyl)benzamide (69).

The ‘HATU coupling’ reaction was performed using 67d (15 mg, 0.05 mmol), 62 (12 mg, 0.05 mmol), HATU (21 mg, 0.056 mmol) and triethylamine (8 mg, 0.077 mmol) to obtain compound 69 as off-white solid (12 mg, yield = 47%). ¹H NMR (500 MHz, CHLOROFORM-d) δ 11.45 (br. s., 1H), 7.94 (d, J = 8.07 Hz, 2H), 7.75 (d, J = 8.07 Hz, 2H), 7.07 - 7.19 (m, 2H), 7.06 (s, 1H), 6.74 - 6.84 (m, 1H), 3.22 (d, J = 7.83 Hz, 2H), 2.97 (br. s., 2H), 2.51 (br. s., 1H), 2.36 (s, 3H), 1.98 - 2.06 (m, 1H), 1.86 - 1.95 (m, 2H), 1.72 - 1.81 (m, 2H). ¹³C NMR (126 MHz, CHLOROFORM-d) δ 163.7, 156.7, 148.8, 140.1, 137.7, 135.5, 135.0, 132.4, 132.3, 128.1, 127.8, 119.6, 118.7, 112.3, 84.9, 70.5, 44.0, 30.4, 25.5, 20.5. MS for C₂₄H₂₃N₆O₃S₂ [M + H]⁺ calculated 507.1268, found 507.1267.

Biology: Cell lines and reagents

The THP1-Blue™ NF-κB cell line was purchased from InvivoGen (San Diego, CA) which contains a stably integrated NF-κB-inducible SEAP. The CellSensor® NF-κB-bla THP-1 cell line was purchased from Thermo Fisher Scientific (Waltham, MA) which contains a stably integrated beta-lactamase reporter gene under the control of the NF-κB response element. QuantiBlue reagent, LPS (lps-eb) and MPLA (vac-MPLA) were purchased from InvivoGen. OVA was purchased from Wothington Biochemical Co. (Lakewood, NJ) and MTT was purchased from Acros Organics. THP-1 cells were purchased from the American Type Culture Collection (Manassas, VA). Primary murine BMDCs were prepared from bone marrow cells harvested from the femurs and tibias of C57BL/6 mice as previously descrived.³⁸

Animals

Seven to nine-week-old wild type BALB/c mice were purchased from The Jackson Laboratories (Bar Harbor, MA). All animal experiments received prior approval from the UCSD Institutional Animal Care and Use Committee.

Measurement of NF-κB activation using THP1-Blue™ NF-κB cells

THP1-Blue™ NF-κB cells were plated in 96-well plates at 10⁵ cells/well in 100 μl RPMI supplemented with 10% fetal bovine serum (FBS, Omega Scientific, Inc., Tarzana, CA), 100 U/mL penicillin, 100 μg/ml streptomycin (Thermo Fisher Scientific) and Normocin (Invivogen). LPS was prepared in assay medium at a concentration of 20 μg/mL Tested compounds were dissolved in DMSO at 1 mM as a stock solution and were further diluted in the LPS solution or media to a final concentration of 10 μM. 100 μL of this solution was then transferred to the plated cells to obtain a final concentration of compound at 5 μM (0.5% DMSO) and LPS at 10 ng/mL or only media for compound alone samples. For dose response activity profiles for compounds, a similar protocol was followed, starting with graded concentrations of compound stocks in DMSO (1 mM, 500 μM, 250 μM, 125 μM and 62.5 μM) and diluting further with LPS to maintain final DMSO concentration at 0.5%. The culture supernatants were harvested after a 20h incubation period. SEAP concentration in the culture supernatants was determined by a colorimetric assay using QuantiBlue (Invivogen). Plate absorbance was read at 630 nm using a Tecan Infinite M200 plate reader (Männedorf, Switzerland). The SEAP concentration was directly proportional to the levels of NF-κB activation, which was 2-point normalized to yield activity for LPS as 100% and activity of compound 1 + LPS as 200%.

Cell viability assay

THP-1 cells were plated in 96-well plates (10⁵ cells/well) in 100 μL RPMI supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/ml streptomycin. Compounds were dissolved in DMSO at 1 mM stock solution and were further diluted to 10 μM in the assay medium. 100 μL of this solution was added to the cells to obtain a final compound concentration of 5 μM (0.5% DMSO). After 18h incubation, a solution of MTT in assay media (0.5 mg/mL) was added to each well and further incubated for 4 to 6 h, followed by addition of cell lysis buffer (15% w/v SDS and 0.12% v/v 12N HCl aqueous solution), incubated overnight, and then absorbance measured at 570 nm using 650 nm as reference using Tecan Infinite M200 plate reader.

Measurement of cytokine induction

THP-1 cells and mouse BMDCs were treated with compound (5 μM) or vehicle (0.5% DMSO) in the presence and absence of LPS (1 ng/mL) overnight. A portion of supernatant was transferred and secreted levels of human IL-8 and murine IL-12 were assayed by ELISA as previously described.³⁸

In vivo adjuvant activity study

BALB/c mice (n=5 per group) were immunized in the gastrocnemius muscle with OVA (20 μg/mice) mixed with MPLA (10 ng/mice) and compound 1, 12d, 18q or 54h (50 nmol/mice) on days 0 and 21. On day 28, immunized mice were bled and OVA-specific IgG titers were measured by ELISA as previously described.³⁹

Statistical analysis

Data are represented as mean ± standard error of the mean (SEM). Origin 7 (Origin Lab, Northampton, MA) graphing software was used for figure preparation while Prism 4 (GraphPad, San Diego, CA) software was used for statistical calculations. One-way ANOVA analysis with Bonferroni’s post hoc testing was used for multiple comparison for in vitro data. For antigen specific antibody data, Bartlett’s one-way ANOVA followed by Dunnett’s post hoc testing was used.

ABBREVIATIONS

ANOVA

Analysis of variance

APC

antigen presenting cells

AS04

Adjuvant System 04

BMDCs

bone marrow-derived dendritic cells

DIPEA

N,N-diisopropylethylamine

DMF

N,N-dimethylformamide

DMSO

dimethyl sulfoxide

EC₅₀

half maximal effective concentration

ELISA

enzyme-linked immunosorbent assay

FBS

fetal bovine serum

FDA

Food and Drug Administration

FRET

Förster resonance energy transfer

HATU

(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

HRMS

high resolution mass spectrometry

HTS

high-throughput screening

IFN

interferon

Ig

immunoglobulin

IL

interleukin

IRAK-M

interleukin-1 receptor-associated kinase-M

LC-MS

liquid chromatography-mass spectrometry

LPS

lipopolysaccharide

m

meta

MPLA

monophosphoryl Lipid A

MTT

(3-[4,5-dimethylthiazol-2-yl]-2,5-dipheyl tetrazolium bromide)

NF-κB

nuclear factor kappa B

NLR

nucleotide-binding oligomerization domain-like receptors (NLRs)

o

ortho

OD

optical density

OVA

ovalbumin

p

para

PRR

pattern recognition receptors

RLR

RIG-I-like receptors

SAR

structure-activity relationship

SEAP

secreted embryonic alkaline phosphatase

THF

tetrahydrofuran

TLC

thin layer chromatography

TLR

Toll-like receptor

TNF

tumor necrosis factor